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Full text of "Xenobiotic effects on gene expression in endometrial cells and placental tissue"

XENOBIOTIC EFFECTS ON GENE EXPRESSION IN ENDOMETRIAL CELLS AND 

PLACENTAL TISSUE 



BY 
GRANTLEY D. CHARLES 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE 

UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS 

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 

1997 



This dissertation is dedicated to the memory of my mother 
VALERIE LOORJN CHARLES 



ACKNOWLEDGMENTS 

I would like to thank first and foremost my mentor, Dr. Kathleen Shiverick, for 
guiding me during the course of my research and for refusing to accept anything less than 
my best. I would also like to thank the present and past members of the laboratory, Dr. 
Phyllis Conliffe, Dr. Scott Masten, Dr. Mary Vaccarello, Dr. Liyan Zhang, Dr. Yara Smit, 
Paul Saunders and most of all Theresa Medrano, for having made this not only a great 
working environment, but a fun and entertaining one as well. 

I would like to express my thanks to my fellow graduate students, Sam, Alan and 
Srini, for their friendship and camaraderie during our study together over the years. I also 
wish to thank the members of my dissertation committee. Dr. William Buhi, Dr. William 
Farmerie, Dr. Jeffrey Harrison, Dr. Thomas Rowe and Dr. David Silverman, for the ready 
access to their advice and support during the course of my work. I am also grateful to the 
remaining faculty and staff of the Department of Pharmacology for having made my time 
here both rewarding and pleasant. 

I would like to acknowledge the following people for the contribution of materials 
required for the completion of this work: Dr. Thomas Rowe, Dr. WiUiam Buhi, Dr. 
WiUiam Greenlee, Dr. Daniel Linzer, Dr. Mary Duckworth and Dr. Michael Soares. 
Especial thanks go to Dr. Maria Grant and Dr. William Farmerie not only for the material 
and support provided during the course of my research, but for the personal guidance and 
instruction they provided. 

Finally, I would like to thank my father, sisters and my uncle and his family for 
their support when the light at the end of the tunnel seemed to distant to attain. 



m 



TABLE OF CONTENTS 

ACKNOWLEDGMENTS iii 

KEY TO ABBREVIATIONS vii 

ABSTRACT ix 

CHAPTER 1: INTRODUCTION 1 

Study Objectives 1 

Uterine Disease: Association with Xenobiotic Agents 2 

Endometriosis: Disease Histogenesis, Etiology and Promotion 5 

Mechanisms of Action of TCDD and B(a)P 7 

Ah Receptor Activity 7 

AhR Independent Mechanisms 8 

Use of Endometrial Carcinoma Cells for the Study of the Potential Role of 

Xenobiotics in Endometriosis 9 

PRL-GH Related Protein Expression in Rodent Placental Tissue 10 

Placental Prolactin Family of Proteins 10 

Biological Activities of Placental Proteins 12 

Angiogenesis, Xenobiotics and Placental-Fetal Development 14 

CHAPTER 2: MATERIALS AND METHODS 16 

Materials 16 

Chemicals and Bioreagents 16 

Recombinant cDNA Clones, Vectors and Plasmids 17 

Antibodies and Antisera Generation 18 

Methods 18 

Cell Culture and Chemical Treatments 18 

RL95-2 Endometrial Carcinoma Cells 18 

Nb2 Cells 19 

Chinese Hamster Ovary (CHO) Ceils 19 

Retinal Endothelial Cells 19 

Expression and Purification of hCAII-PLP-C Fusion Protein 20 

Construction of the pET22b(+)PLP-C Bacterial Expression System.21 

Expression Vector 21 

Purification of Recombinant His-PLP-C 21 

Amino-terminal Sequence Analysis 22 

Construction of PLP-B Mammalian Expression System 22 

Expression Vector 22 

Transfection and Stable Mammalian Expression of PLP-B .. 23 

Basal Zone Explant Conditioned Media 24 

Immuno-Affinity Protein Purification 24 

Endothelial Cell Migration Assay 25 

In Vitro Invasion Assay 25 

Nb2 Assay for Lactogenic Activity 26 



IV 



EGFR Binding Assay 27 

Western Immunoblot Analysis 27 

General Proceure 27 

EOF Receptor and CYPlAl Protein 28 

PLP-B and PLP-C Protein 28 

RNA Isolation & Northern Blot Analysis 28 

Nuclear Run Off Assay 29 

Fibrin Zymography 30 

Data Analysis 31 

CHAPTER 3: EFFECTS OF TCDD AND B(a)P ON CELLULAR 
PROLIFERATION AND EOF RECEPTOR EXPRESSION 

IN THE RL95-2 CELL LINE 32 

Introduction 32 

Results 33 

Effects of TCDD and B(a)P on CYPlAl and CYPIBI 

mRNA in RL95-2 Cells 33 

Effects of TCDD on "^I-EGF Binding in RL95-2 Cells 34 

Effects of TCDD and B(a)P on EGFR and CYPlAl 

Protein in RL95-2 Cells 34 

Effects of TCDD and B(a)P on Cellular Proliferation 

in RL95-2 Cells 35 

Effect of TCDD and B(a)P on Steady State c-myc 

mRNA Levels 36 

Discussion 36 

CHAPTER 4: EFFECTS OF TCDD AND B(a)P ON CELLULAR 
INVASIVENESS AND THE EXPRESSION OF uPA AND 

TIMPs IN A HUMAN ENDOMETRIAL CELL LINE 47 

Introduction 47 

Results 49 

Evaluation of the Effect of TCDD and B(a)P on 

RL95-2 Cellular Invasiveness 49 

Effect of TCDD and B(a)P on uPA mRNA SteadyState 

Levels and Plasminogen Activity in RL95-2 Cells ... 50 
Effects of TCDD and B(a)P on TIMP mRNA Expression 

in RL95-2 Cells 51 

Discussion 51 

CHAPTER 5: EFFECTS OF TCDD ON IL-lp AND TNFa IN A HUMAN 

ENDOMETRIAL CELL LINE 66 

Introduction 66 

Results 67 

Effects of TCDD on IL-ip and TNF-a mRNA levels 

in RL95-2 Cells 67 

Effect of TCDD on the Rate of Transcription of CYPlAl, 

CYPIBI, uPA, and IL-ip mRNA in RL95-2 Cells ..68 
Discussion 69 



CHAPTER 6: EXPRESSION AND PURIFICATION OF RECOMBINANT 

AND NATIVE PROLACTIN-LIKE PROTEINS B AND C 78 

Introduction 78 

Results 79 

Purification of Recombinant hCAII-PLP-C 79 

Recombinant Expression and Purification of 

pET22b(+)His-PLP-C 81 

Expression of Recombinant PLP-B 82 

Purification and Western Immunoblot Analysis of Native 

PLP-B and PLP-C 83 

Discussion 84 

CHAPTER 7: CHARACTERIZATION OF NATIVE AND RECOMBINANT 

PROLACTIN-LIKE PROTEINS B AND C 100 

Introduction 100 

Results 101 

Effects of Native PLP-B and PLP-C and Recombinant 

PLP-C on Nb2 Lymphoma Cell Proliferation 101 

Effects of Placental Conditioned Medium, and Native 

PLP-B and PLP-C on Endothelial Cell Migration .... 101 
Discussion 102 

CHAPTER 8 : CONCLUSIONS AND FUTURE DIRECTIONS Ill 

LIST OF REFERENCES 117 

BIOGRAPHICAL SKETCH 136 



VI 



KEY TO ABBREVIATIONS 



All 

AhR 

Amt 

B(a)P 

BEE 

PNF 

BSA 

c-myc 

cDNA 

CHX 

CIAP 

CYPlAl 

CYPIBI 

DDT 

DES 

DIM 

DMEM 

DMSO 

DNA 

DRE/XRE 

DTT 

ECM 



aryl hydrocarbon 

aryl hydrocarbon receptor 

aryl hydrocarbon nuclear translocator 

benzo(a)pyrene 

bovine brain endothelial 

P-napthoflavone 

bovine serum albumin 

cellular myc ribonucleic acid 

complementary deoxyribonucleic acid 

cycloheximide 

calf intestinal alkaline phosphatase 

cytochrome P450 lAl 

cytochrome P450 IBl 

dichlorodiphenyltrichloroethane 

diethylstilbestrol 

3,3'-diindolylmethane 

dulbecco's modified eagle's medium 

dimethyl sulfoxide 

deoxyribonucleic acid 

dioxin responsive element/xenobiotic responsive element 

dithiothreitol 

extracellular matrix 



Vll 



EGF 

FBS 

GH 

GnRH 

hCAE 

hr 

hsp 

IC3 

IGF-II/M6P 

lUGR 

kb 

kDa 

3-MC 

MMP 

mRNA 

MTX 

MXC 

MW 

NLS 

PAH 

pAMBS 

PBS 

PCB 

4-PeCDF 

PKC 

PL 



epidermal growth factor 

fetal bovine serum 

growth hormone 

gonadotropin releasing hormone 

human carbonic anhydrase II 

hours 

heat shock protein 

indole-3-carbinol 

insulin-like growth factorII/mannose-6-phosphate 

polyaromatic hydrocarbon 

kilobases 

kilodaltons 

3-methylcholanthrene 

matrix metalloproteinase 

messenger ribonucleic acid 

methotrexate, aminopterin 

methoxychlor 

molecular weight 

N-lauroylsarcosine 

polyaromatic hydrocarbon 

p-aminomethylbenzenesulfonamide 
phosphate-buffered saline 
polychlorinated biphenyl 
2,3,4,7, 8-pentachlorodibenzofuran 
protein kinase C 
placental lactogen 



vm 



PLF 

PLP 

PMSF 

PRL 

PRP 

RNA 

SEM 

SDS-PAGE 

TCDD 

TeCB 

TGF 

TIMP 

TNF-a 

ffA 

uPA 



proliferin 

prolactin-like protein 

phenyl-methyl sulfonyl fluoride 

prolactin 

proliferin-related protein 

ribonucleic acid 

standard error of mean 

sodium dodecyl sulfate polyacrylamide gel electrophoresis 

2,3,7, 8-tetrachlorodibenzo-p-dioxin 

tetrachlorobiphenyl 

transforming growth factor 

tissue inhibitor of metalloproteinase 

tumor necrosis factor alpha 

tissue plasminogen activator 

urokinase plasminogen activator 



IX 



Abstract of Dissertation Presented to the Graduate School of 
the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 



XENOBIOTIC EFFECTS ON GENE EXPRESSION IN ENDOMETRIAL CELLS AND 

PLACENTAL TISSUE 

By 

Grantley D. Charles 
December 1997 



Chairman: Kathleen T. Shiverick 

Major Department: Pharmacology and Therapeutics 

This study evaluated, firstly, the potential role of the environmental contaminants 
2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene [B(a)P] in uterine disease 
utilizing an endometrial adenocarcinoma cell line RL95-2. Secondly, this research study 
investigated the potential lactogenic and angiogenic activity of two members of the 
prolactin/growth hormone family, prolactin-like proteins B and C which are secreted by the 
rat placenta during pregnancy. 

TCDD and B(a)P were evaluated for their abihty to alter the expression of growth 
factor and cytokine genes including epidermal growth factor (EGF) receptor, urokinase 
plasminogen activator (uPA), interleukin (EL-lp) and tumor necrosis factor (TNF-a). This 
study demonstrated that both TCDD and B(a)P induced the expression of CYPlAl in 
RL95-2 cells, but only B(a)P significantly decreased EGF receptor expression. TCDD but 
not B(a)P significantly increased the steady state level of uPA messenger ribonucleic acid 
(mRNA), however neither chemical was able to significantly alter the associated fibrinolytic 
activity of conditioned medium from treated RL95-2 cultures. Furthermore, TCDD 



increased the mRNA expression level of TNF-a in a time-dependent and IL-1 p in a time- 
and dose-dependent manner. Finally, B(a)P, but not TCDD, was able to inhibit 
significantly, the overall proliferation and invasiveness of these endometrial 
adenocarcinoma cultures. These results indicate that TCDD and B(a)P can alter the gene 
expression of members of the growth factor/cytokine network in uterine tissue and so 
potentially contribute to the promotion of uterine disease. 

Native preparations of PLP-B and PLP-C purified from the conditioned medium of 
placental explant cultures exhibited little lactogenic activity relative to ovine prolactin, as 
evaluated by their ability to stimulate the proliferation of the rat Nb2 lymphoma cell line. 
Conditioned media from gestation day 18 rat placenta significantly increased the directional 
migration of human retinal endothelial cells, a measure of angiogenic activity. In contrast, 
neither immunopurified native PLP-B nor PLP-C proteins showed any significant 
stimulation of endothelial cell migration. Both PLP-B and PLP-C were successfully 
expressed as recombinant proteins in mammalian and bacterial systems. Recombinant 
PLP-C, in contrast to the native preparation, did not exhibit any measurable lactogenic 
activity. In summary, the angiogenic activity exhibited by conditioned medium of rat 
placental cultures is not associated with either PLP-B or PLP-C. 



XI 



CHAPTER 1 
INTRODUCTION 



Study Objectives 



This research was undertaken to investigate the potential role of environmental 
contaminants in uterine disease and altered placental function. The objectives of the 
projects were to investigate the potential mechanisms whereby prototype xenobiotics might 
(a) potentially contribute to the promotion of uterine disease in general, and endometriosis 
in particular, as well as (b) alter placentaL/fetal growth and development. Firstly, recent 
evidence indicates that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other 
environmental pollutants such as methoxychlor (MXC) may contribute to the promotion of 
uterine endometriosis, a benign proliferative disorder (Cummings and Metcalfe, 1995; 
Gerhard and Runnebaum, 1992; Koninckx et al., 1994; Mayani et al., 1997; Rier et al., 
1993). It is now recognized that TCDD and other xenobiotics, which are capable of 
binding to a cytosolic receptor designated the arylhydrocarbon receptor (AhR), can also act 
as endocrine and growth modulators via alterations in the expression of a number of genes. 
Endometriosis is an enigmatic disorder, however, with little being known regarding its 
histogenesis and maintainence. Our hypothesis for this investigation is that TCDD and 
benzo(a)pyrene B(a)P are able to alter the expression of uterine genes and gene products 
involved in implantation and immune responsiveness which can contribute to uterine 
disease etiology. 

Secondly, our laboratory has previously shown that gestational exposure of 
pregnant rats to |3-napthoflavone (pNF) and 3-methylcholanthrene (3-MC), prototypical 



polyaromatic hydrocarbons (PAH) and AhR ligands, resulted in intrauterine fetal growth 
retardation (lUGR) and decreased placental function (Fuhrman-Lane et al., 1983; Shiverick 
et al., 1984). Furthermore, PNF treatment was found to be associated with the decreased 
secretion of a family of placental prolactin-like proteins (PLPs) into the conditioned media 
of placental explant cultures (Shiverick et al., 1991). These rat placental prolactin-like 
proteins have become of particular interest based upon the recent demonstration of the 
angiogenic and angiolytic activity of two potentially homologous proteins in murine 
placenta (Jackson et al., 1994). Thus, these data led us to hypothesize that pNF-mediated 
alteration in the expression of rat placental PLPs could result in a decreased ability of the 
placenta to develop a vascular network capable of maintaining adequate fetal growth. To 
investigate this possibility, we purified native rat placental PLP-B and PLP-C as well as 
expressed recombinant forms of these proteins in bacterial and mammalian systems in order 
to evaluate them for potential lactogenic and angiogenic activity. The objective of this part 
of my reseai-ch has been to elucidate the potential role(s) of these placental members of the 
PRL-GH family during the course of pregnancy. 

Uterine Disease: Association with Xenobiotic Agents 

There has been increasing public and scientific concern that environmental 
pollutants may be able to disrupt the normal hormonal milieu in humans and animals 
leading to disease pathologies. Many naturally occurring and man-made chemicals present 
in the environment possess estrogenic and antiestrogenic activity. These include plant and 
fungal products, pesticides, plasticizers, and other industrial and agricultural chemicals 
(Stancel et al., 1995). Reports of abnormal sexual development in reptiles (Guillette et al., 
1994) and birds (Fry, 1995) have provided evidence to support the proposal that select 
environmental chemicals function as endocrine modulators. Concerns continue to grow 
regarding the potential involvement of these agents in such reproductive abnormahties as 



breast cancer, endometriosis, fibroids and uterine adenocarcinoma in women (Newbold, 
1995). 

The growth response of the uterus to steroid hormones is a highly regulated process 
in reproductively cycling women. Hence this is a potential site for the manifestation of the 
endocrine dismptive effects of environmental pollutants. Studies in rodents exposed to 
diethylstilbestrol (DES), a potent synthetic estrogen, during the prenatal and neonatal 
period showed epithelial and stromal stimulation of the uterine horns, cystic endometrial 
hyperplasia, as well as a low incidence of benign (leiomyoma) and malignant 
(adenocarcinoma) uterine tumors (Newbold et al., 1990; Newbold, 1995). This evidence 
supported the conclusion that developmental exposure to estrogen affects the pattem of 
uterine cell differentiation, resulting in later morphological and neoplastic alterations. 

A number of environmental pollutants have been shown to modulate the growth and 
differentiation of uterine tissue in both in vitro and in vivo models. The pesticides 
dichlorodiphenyltrichloroethane (DDT) and MXC stimulated DNA synthesis in primary 
cultures of uterine epithehal and stromal cells respectively (Tiemann et al., 1996). The 
administration of o,p'-DDT to immature rats mimicked the effects of estrogen by increasing 
uterine weight, as well as by stimulating DNA synthesis and cell division which led to 
hyperplasia in the uterine luminal epithelium, stroma and myometrium (Kupfer, 1981; 
Robison et al., 1985). Similarly, the polychlorinatedbiphenyl (PCB) Arochlor 1221 was 
found to induce uterine growth in neonatally exposed rats (Gellert, 1978). 

Some environmental agents, however, exhibit activity which opposes the effects of 
estrogen. The characteristic example is TCDD which has been demonstrated to have 
antiestrogenic properties. TCDD exposure has been linked with a variety of antiestrogenic 
responses in the female rat uterus including inhibition of constitutive and 17j3-estradiol- 
induced uterine wet weight increase, diminished nuclear and cytoplasmic estrogen and 
progesterone receptor levels, as well as decreased epidermal growth factor (EOF) receptor 
binding, EGF receptor and EGF receptor mRNA levels, and c-fos proto-oncogene mRNA 



levels (Astroff et al., 1990, 1991; Romkes et al., 1987, Romkes and Safe, 1988). TCDD 
has also been associated with a decreased age-related incidence of tumors of the uterus 
(Kochiba et al., 1978). It should be noted, however, that low dose, short-term 
admistration of TCDD did not appear to alter the uterotrophic response to exogenous 
estrogen in ovariectomized rats (Shiverick and Muther, 1982). The PCB congener 
3,3',4,4'-TeCB was reported to antagonize the uterotrophic effects of estradiol in the 
immature female Sprague-Dawley rat (Jansen et al., 1993), while neonatal exposure of 
female Wistar rats to B(a)P was found to significantiy reduce uterine estrogen receptor 
density in adulthood (Csaba and Inczefi-Gonda, 1993). 

It has been hypothesized that these environmental pollutants could potentially 
contribute to an increased incidence of uterine disease in the female population based upon 
their ability to modulate the growth and differentiation of uterine tissue. Evidence from 
epidemiological and laboratory studies is beginning to implicate a number of these 
environmental toxicants in the etiology of uterine disease pathologies. For example, 
epidemiological data indicates that cigarette smoking is linked with a modest decrease in 
risk for endometrial hyperplasia, while smoking women have a risk for endometrial cancer 
less than half that of non-smokers, an effect more pronounced in postmenopausal 
compared with premenopausal women (Baron et al., 1990; Baron, 1996). Epidemiological 
data also suggest that there is an inverse association of cigarette smoking with uterine 
fibroids and endometriosis (Baron, 1996; Cramer et al., 1986; Matorras et al., 1995). 
There is presently insufficient data, however, to support a conclusion regarding the effects 
of organochlorine compounds on endometrial cancer (Ahlborg et al., 1995). 

Recent studies in rhesus monkeys and rodents indicate that TCDD and MXC may 
act as promoters in the development of endometriosis (Cummings et al., 1996; Cummings 
and Metcalfe, 1995; Rier et al., 1993). In rhesus monkeys, dietary intake of TCDD was 
associated with an increased incidence and severity of endometriotic lesions (Rier et al., 
1993). In surgically-induced rodent models of endometriosis, both TCDD and MXC 



supported the development and growth of the endometriotic implants (Cummings et al., 
1996; Cummings and Metcalfe, 1995). In addition, human epidemiological evidence is 
accumulating to support an association between TCDD and the promotion of uterine disease 
(Koninckx et al., 1994; Mayani et al., 1997). Women with endometriosis were also 
reported to have increased concentrations of PCBs in their blood (Gerhard and 
Runnebaum, 1992). Experiments in rhesus monkeys with the PCB Arochlor 1254, 
however, concluded that the incidence and severity of endometriotic lesions observed in the 
animals did not have any relation to the doses of PCB ingested during the study (Arnold et 
al., 1996). Thus, the data are not conclusive and may reflect the different structure of 
organochlorine compounds as well as their routes of metabolism. 

Endometriosis: Disease Histogenesis. Etiology and Promotion 

Endometriosis, the term first coined by Sampson (1921), is defined as the presence 
of glands and stromal tissue, histologically similar to endometrium, outside the uterine 
cavity and myometrium which is associated with vascularization and cellular proliferation. 
The most common location of endometriosis is in the pelvis with greatest frequency 
occurring in the ovaiy (Jenkins et al., 1986). The symptoms usually observed include the 
presence of pelvic masses, pain, and infertility (Barbieri, 1992; Olive and Schwartz, 1993). 
In fact, a recent study indicates that 80% of laparoscopics for infertihty result in diagnosis 
of this disease (Thomas and Prenfiss, 1992), leading to 400,000 hysterectomies being 
performed in the U.S.A in 1984 (Natl. Cen. Health Stat. 1986). Although the exact 
incidence has been difficult to determine due to the asyptomatic nature of the disease which 
requires laparoscopy or surgical visualization for a definitive diagnosis, a prevalence of 
10% has been estimated in the general female population (Olive and Schwartz, 1993). 

Evidence from epidemiological and cHnical studies, as well as work on animal 
disease models, suggests that environmental agents as well as other factors may have a 
significant role to play in the etiology and pathogenesis of the disease (Cramer et al., 1986; 



Gerhard and Runnenbaum, 1992; Koninckx et al., 1994; Matorras et al., 1995; Mayani et 
al., 1997; Rier et al., 1993). The main etiological factors appear to be exposure to estrogen 
and the process of cychc shedding of the uterine lining. In this regard, endometriosis is 
more common in women with short menstrual cycle lengths (< 27 days) and longer flow 
periods (> 1 week) (Cramer et al, 1986; Matorras et al., 1995), while occurring 
infrequently before puberty or after menopause. These studies further reported that 
endometriosis was less common in smokers and in women with a low body mass index, 
both factors which lead to lower endogenous estrogen stimulation. Recently, exposure to 
environmental contaminants like TCDD and PCBs have also been associated with an 
increased disease incidence in animal and human studies (Gerhard and Runnenbaum, 1992; 
Mayani et al., 1997; Rier et al., 1993). 

The physiological and molecular mechanisms whereby endometriotic tissue 
develops and persists outside the uterine cavity are poorly understood and remain 
controversial. Endometriosis has been described as the disease of theories, there being 
several existing hypotheses as to its pathogenesis. Three main concepts predominate (as 
reviewed in van der Linden, 1996; OUve and Schwartz, 1993). The in situ development 
theory states that endometriosis develops in the location where it is found, thought to be the 
consequence of metaplasia of peritoneal or ovarian tissue (Lauchlan, 1972). This theory 
does not, however, explain why the disease occurs exclusively in women, in the pelvic 
organs, during their reproductive years. The second concept, the induction theory, argues 
that endometriosis results from the differentiation of mesenchyme induced by substances 
released by degenerating endometrium (Merrill, 1966). The third theory of histogenesis is 
that uterine endometrium is transplanted to ectopic locations through lymphatic and vascular 
dissemination and retrograde menstruation (Sampson, 1927). The implantation theory is at 
present the most widely held with the anatomical patterns of the disease being consistent 
with retrograde menstruation (Jenkins et al., 1986). The implantation theory, however, 
does not account for the fact that retrograde menstruation occurs in the vast majority of the 



female population, whereas endometriosis develops in only a fraction of the these women. 
This inconsistency has led to the postulate that an impaired immune response or response to 
tissue injui-y may result in the inability to remove refluxed menstrual debris, thereby 
increasing the possibility of endometriosis (Dmowski et al., 1994; Gleicher and Pratt, 
1993). 

Mechanisms of Action of TCDD and B("a)P 

Ah Receptor Activity 

The aryl hydrocarbon receptor (AhR) mediates most of the toxicological effects of 
certain halogenated aromatic hydrocarbons that are widely disseminated in the environment, 
including TCDD and polyaromatic hydrocarbons (PAHs) found in cigarette smoke and 
industrial exhaust (Hankinson, 1994; Whitlock, 1993). The unUganded AhR is a basic- 
heUx-loop-hehx protein which resides in the cytoplasm in association with the 90 kDa heat 
shock protein (hsp90) and functions as a ligand-activated transcription factor (Hankinson, 
1994; Whitlock et al., 1996). While TCDD and related PAHs like B(a)P are known to bind 
the receptor, little is known regarding the binding of physiological ligands to the Ah 
receptor. Compounds like indole-3-carbinol (IC3) and 3,3'-diindolylmethane (DIM) found 
in cruciferous vegetables, however, have been shown to activate the AhR, although they 
possess a much lower receptor affinity (Jellinck et al., 1993; Kleman et al., 1994). The 
AhR has been implicated as the primary mediator of toxicity of the PAHs based on 
evidence that the toxicity of individual congeners is correlated with affinity for the receptor, 
as well as that susceptibility to a range of toxic effects segregates with the Ah allele in 
highly responsive mouse strains like C57BL/6 (Okey et al., 1994). 

The binding of ligands like TCDD is thought to result in allosteric changes in the 
dimerization domain of the AhR which allows for release of the hsp90, followed by 
translocation to the nucleus and interaction with a nuclear protein, the arylhydrocarbon 
nuclear translocator (Arnt). There is some evidence which implicates protein kinase C 



8 

(PKC) and tyrosine kinase in the generation of an active AiiR/Arnt complex, yet the precise 
role of these kinases remains unclear (Gradin et al., 1994; Schafer et al., 1993). The 
heterodimeric AhR:Amt complex, possibly in association with other proteins/factors (Chan 
et al., 1994; Dunn II et al., 1996), is able to bind to enhancer sequences termed dioxin or 
xenobiotic response elements (DREs or XREs) upstream of structural genes such as 
CYPIAI. Xenobiotic-induced binding of the AhR/Amt complex to enhancer chromatin 
sequences is then associated with localized changes in chromatin structure manifested by 
increased accessibility of the gene promoter DNA sequence to general transcription factors 
such as the TATA-binding protein. The initiation complex formed at the promoter then 
allows for gene transcription to be initiated (Okino and Whitlock, 1995; Whitlock et al., 
1996). 

The principal route of AhR mediated toxicity is thought to occur via the ability of 
the AhR/Amt complex to facihtate the increased transcription of metabolic enzymes like 
CYPlAl leading to the bioactivation of pretoxicants to their reactive metabolites, which 
may result in cytotoxic, carcinogenic or teratogenic effects. In addition, these regulated 
enzymes may also play a role in the metabolism of endogenous compounds involved in the 
control of cellular growth and differentiation (Hankinson, 1994; Okey et al., 1994). The 
fact that TCDD, B(a)P and related compounds have been demonstrated to alter gene 
expression in uterine tissue, coupled with the ubiquity of AhR tissue expression (Dolwick 
et al., 1993), makes it feasible to investigate the potential action of these compounds in the 
promotion of uterine pathologies. 

AhR Independent Mechanisms 

Some scientists question that AhR-mediated induction of gene transcription is the 
sole route for the toxic effects of TCDD and related compounds. Recent work under cell- 
free conditions has demonstrated that TCDD is able to activate protein kinases in the 
absence of a nucleus (Enan and Matsumura, 1995). Induction of immediate-early response 



protooncogenes like c-fos andjwnB by TCDD and B(a)P appears to be independent of AhR 
and Arnt in variant hepatoma cell lines (Hoffer et al., 1996; Puga et al., 1992). This 
evidence lends support to the possibility of distinct signal transduction pathways for the 
mediation of the toxic effects of these compounds. Furthermore, the toxic effects of B(a)P 
may be exacerbated by metabohsm in mammalian cells to reactive products that can 
covalently bind to DNA, as well as through the generation of reactive oxygen species 
capable of producing direct cellular damage (Leadon et al., 1988). TCDD is itself highly 
resistant to metabolism and has an approximate 7-10 year half life in humans. These 
differences in mechanisms of action may account for the observed differences in the 
epidemiological findings of cigarette smoking compared with TCDD as relates to uterine 
disease (Ahlborg et al., 1995). Our study of their comparative effects in our endometrial 
culture system should further the understanding of these empirical observations. 

Use of Endometrial Carcinoma Cells for the Study of the Potential Role of Xenobintics in 

Endometriosis 

The question sometimes arises as to the appropriateness of utilizing in vitro cultures of 
transformed cells as models for the study of disease pathologies. In this regard, a major 
concern is the potential difference in responsiveness of transformed cells compared to the 
original uterine tissue. The use of primary endometrial cell culture, however, presents a 
number of problems, such as the cyclical hormonal variation which exists throughout the 
menstiual cycle, the lack of tissue homogeneity, finite life span, interindividual variation of 
the tissue, and potential loss of steroid and other receptor signalling systems (Tabibzadeh et 
al., 1990; Watson et al., 1994). The use of tissue from patients with endometriosis 
presents similar difficulties with patient variabihty in severity of this disease, as well as that 
of the availability of a reliable supply of tissue for culture maintainence. 



10 

The endometrial adrenocarcinoma RL95-2 cell line was established by Way et al 
(1983) and has been investigated with respect to the action of several growth factors 
including EGF, TGFa and TGF|3 (Korc et al., 1986, 1987). Liu and Teng (1994) 
demonstrated that estrogen was able to produce measurable responses in the RL95-2 cell 
line. Similarly, Grenman et al (1988) demonstrated RL95-2 responsiveness to both 
estrogen and progesterone. RL95-2 also exhibits significant EGF and TGFp binding 
activity (Dumontet al., 1995; Korc et al., 1986; Lelle et al., 1993). Endometriotic tissue 
from patients transcends the clinico-pathologic distinction between a benign disease and an 
invasive neoplasm. While histologicaly benign, endometriotic tissue invades local pelvic 
structures (Koninckx and Martin, 1992). Hence we considered the use of the 
adenocarcinoma RL95-2 cell line to be appropriate as an in vitro model for the purposes of 
the present study. 

I PRL/GH Related Rrotein Expression in Rodent Placental Tissue 

Placental Prolactin Family of Proteins 

The rodent develops two distinct placental structures during the course of gestation. 
The first to develop is the choriovitelline placenta which disappears by day 14 of gestation. 
While the chorioviteUine placenta is degenerating, the chorioallantoic placenta comes into 
existence being composed of three major regions, the labyrinth (60%), the basal or 
junctional zone (15%), and the decidua basalis, subplacental region and metrial gland 
(25%) which comprise the remaining part of the total placental structure late in gestation 
(Davies and Glasser, 1968). The rodent placenta is a rich source of placental lactogens 
(PLs) and several other proteins which have a structural homology to pituitary PRL rather 
than to GH (Soares et al., 1991; Southard and Talamantes, 1991). It is predominantly in 
the basal or junctional zone that the PRL-like proteins are expressed. 



11 

Amino acid or nucleotide sequence data is now available for fifteen of these 
proteins. The seven placental lactogens include rat and mouse PL-I (Colosi et al., 1987a; 
Robertson et al., 1990), rat PL-Iv (Deb et al., 1991; Robertson et al., 1991), rat PL-I 
mosiac (Hirosawa et al., 1994), hamster, rat and mouse PL-II (Duckworth et al., 1986a; 
Jackson et al., 1986; Southard et al., 1986). The seven prolactin-like proteins include: 
mouse Proliferin (PLF) and Proliferin-Related Protein (PRP) (Linzer and Nathans, 1985; 
Linzer et al, 1984), and rat prolactin-like proteins A, B, C, Cv and D (Dai et al., 1996; 
Deb et al., 1991c; Duckworth et al., 1986b; Duckworth et al., 1988; Iwatsuki et al., 1996). 
Rat decidual prolactin-related protein (dPRP), which shows a high degree of sequence 
identity to PLP-C, has also been cloned from rat decidua (Roby et al., 1993), while a PRL- 
like cDNA from the midgestation hamster placenta similar to PL-I has recently been isolated 
and characterized (Barnes and Renegar, 1996). These proteins exhibit both cell and 
temporal specific patterns of expression, being secreted predominantly by the 
spongiotrophoblast and trophoblast giant cells of the junctional or basal zone. 

Comparison of the amino acid sequences of these proteins indicates that amongst 
the PL-Is there is over 70% sequence identity and sinndlar homology exists among the PL- 
IIs. In contrast, the prolactin-like proteins A, B, C, Cv, D, Prohferin (PLF) and Prohferin 
Related Protein (PRP) have between 12-40% sequence identity with each other and to the 
PLs (Iwatsuki et al., 1996; Southard and Talamantes, 1991). The structural assignment of 
these proteins to the PRL family has been based on the positioning of conserved cysteine 
residues, as well as additional amino acid sequence homologies. Any similarities in 
biological activities to PRL were not considered a requirement for inclusion, since in most 
cases these activities were yet to be determined. Therefore, as a group they bear at 
maximum a 45% sequence homology to pituitary PRL. 

Prolactin is known to exhibit a broad range of distinct physiological actions 
important in reproduction. These include regulation of amniotic fluid volume and ion 
content, development of the mammary gland and milk protein production, along with 



12 

suppressing the immune response and uterine contractility (Handwerger et al., 1992). At 
the onset of pregnancy, pituitary PRL secretion exhibits a twice daily surge which abruptly 
terminates at midgestation when the chorioallantoic placenta develops and secretion remains 
depressed for the rest of gestation (Smith and Neill, 1976). The presence of pituitary PRL 
after day 6 of gestation is not required for the maintenance of the corpus luteum (Morishige 
and Rothchild, 1974). It has also been observed that removal of the anterior pituitary in the 
rat after midgestation did not interupt pregnancy (Pencharz and Long, 1931). Pituitary 
PRL could not be replaced by decidual PRL in rodents, as it may be in humans, since 
rodent decidua does not express this hormone (Handwerger et al., 1984). The secretion by 
the placenta of high levels of placental lactogens and prolactin-related proteins during the 
course of pregnancy has led to the hypothesis that they are of importance in the placental- 
fetal growth axis. 

Biological Activities of Placental Proteins 

There has been a great deal of interest in determining whether or not these newly 
discovered proteins were "PRL-like" in their ability to bind the PRL receptor, or in the 
expression of similar bioactivities. Studies characterizing these rodent placental proteins 
have demonstrated that some of them do in fact exhibit PRL-like activities. This has been 
shown by the abihty of haPL-II, rat and mouse PL-I and PL-II and rat PL-Iv to exhibit 
lactogenic activity by stimulating prolactin-like responses in rat Nb2 lymphoma cells 
(Cohick et al., 1995; Colosi et al., 1987a, b; Deb et al., 1991c; Robertson et al., 1982, 
1994), in mammary gland epitheUal cell differentiation (Soares et al., 1983; Southard et al., 
1986), and in the pigeon crop sac assay (Colosi et al., 1982). Most of the other prolactin- 
like members of the family have all been expressed, but evidence does not show lactogenic 
activity (Cohick et al., 1997; Conhffe et al., 1994; Rasmussen et al., 1996). Mouse PL-1 
and PL-II have also recendy been shown to be luteotropic and to support progesterone 
production in the mouse at midgestation (Galosy and Talamantes, 1995). The daily surges 



-1 



13 

of pituitary PRL have been demonstrated to be indirectly inhibited by placental lactogen 
(Tomoganeet al., 1992). The hypothesis thus arose that this fanoily of secreted placental 
members of the PRL-GH family could somehow, perhaps via a feedback mechanism, 
replace PRL functionally during gestation. 

Two members of the placental PRL-GH family, proliferin (PLF) and proliferin 
related protein (PRP), have recently been shown to display angiogenic and angiolytic 
activity, respectively (Jackson et al, 1994). PLF and PRP have also been shown to 
compete with 16K PRL for binding to membranes of bovine brain endothelial (BBE) cells 
(Clapp and Weiner, 1992). The 14K and 16K forms of PRL, enzymatically generated N- 
terminal fragments of 23K pituitary PRL, are themselves potent angiolytic factors in BBE 
ceUs (Clapp et al., 1993, 1994; Ferrara et al., 1991). Furthermore, PLF has been 
demonstrated to bind to capillary endothelial cells in the placenta (Jackson et al., 1994), as 
well as to sites in the developing embryonic vertebral and vascular structures (Jackson and 
Linzer, 1997). Competition and comparative binding studies indicate that the insulin-like 
growth factorIFmannose-6-phosphate receptor is involved in PLF binding (Lee and 
Nathans, 1988; Volpert et al., 1996), as well as to a receptor in uterine membrane 
preparations of pregnant mice (Nelson et al., 1995). The chemotaxis initiated by PLF and 
mediated by the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor appears to occur 
through a G protein-coupled pathway via MAPK activation. This was demonstrated by the 
fact that PLF stimulated MAPK activity and endothelial cell chemotaxis, both activities 
being blocked by pertussis toxin and the specific inhibitor of MAPK kinase, PD 098059 
(Groskopf et al., 1997). Although PLP-A, PLP-B, PLP-C and dPRP have been expressed 



I as recombinant proteins, their biological role(s) remain to be elucidated (Cohick et al., 

; 

j 1997; Conhffe et al., 1994; Deb et al., 1993; Rasmussen et al., 1996). 

Our laboratory has previously characterized the expression of a number of these 
" placental prolactin-like proteins in the rat placenta (Ogilvie et al., 1990a, b). Observations 

from our laboratory have associated matemal xenobiotic exposure and protein malnutrition 

I 



14 

with the decreased secretion of placental prolactin-like proteins from basal zone explant 
cultures (Conliffe et al., 1995; Shiverick et al., 1991). A further correlation was noted 
with decreased placental vascularization and intrauterine growth retardation which has led 
us to further investigate the potential for two of these proteins, PLP-B and PLP-C, to 
exhibit lactogenic and angiogenic activity. Although they may not be the homologs of PLF 
and PRP, they may function similarly, although they appear to lack the conserved amino 
acid residues essential for lactogenic activity (de Vos et al., 1992; Goffin et al., 1993, 
1994, 1996; Somers et al., 1994; Southard and Talamantes, 1991). Angiogenesis is an 
important factor in placental development. Placental vascular growth begins early in 
pregnancy and continues throughout gestation, with dramatic increases in fetal-maternal 
blood flow. Similarly, placental anti-angiogenic factors likely target the maternal placental 
vasculature and may function to limit vascular development and possible invasion by fetal 
tissue. Therefore the potential for PLP-B and PLP-C to act as angiogenic or angiolytic 
factors warrants investigation. 

Angiogenesis. Xenobiotics and Placental-Fetal Development 

Angiogenesis was first coined as a term to describe the formation of new blood 
vessels in the placenta (Hertig, 1935). It is the biological mechanism of new capillary 
formation involving the activation, migration and proliferation of endothelial cells from pre- 
existing venules (Hockel et al., 1993). Defects in angiogenesis may contribute to a variety 
of disorders such as endometrial hyperplasia, dysfunctional uterine bleeding, 
endometriosis, pregnancy loss, pre-eclampsia and cancer (Gordon et al., 1995). The 
process of angiogenesis has a critical role to play in placental/fetal establishment and 
development (Reynolds et al., 1992; Welsh and Enders, 1991). Throughout gestation, 
placental transport capacity keeps pace with fetal growth and uterine blood flow increases 
approximately three to four-fold from mid to late gestation (Reynolds et al., 1986). 
Consequently, factors which contribute to inadequate placental vascular development may 



15 

have a tremendous impact on fetal growth and development, and, ultimately, on neonatal 
growth and survival. Embryonic wastage and reduced birth weights are recognized to be 
major socio-economic problems associated with pregnancy (Reynolds and Redmer, 1995). 

The association of environmental agents like cigarette smoke, TCDD and PCBs 
with the incidence of teratogenicity, low birth weight and fetotoxicity (Couture et al., 1990; 
McNulty, 1985; Sachs, 1989) may be related to their ability to interfere with the 
neovascularization process during the course of gestation. Evidence to support this 
hypothesis has begun to accumulate. For example, pregnant mice exposed to TCDD and 
2,3,4,7, 8-pentachlorodibenzofuran (4-PeCDF) exhibit rupture of the embryo-maternal 
vascular barrier and the visceral yolk sac membrane, resulting in the hemorrhaging of 
embryonic blood into the maternal circulation and the uterine and amniotic cavities (Khera, 
1992). Similarly, TCDD exposure in bird and fish models leads to pericardial and yolk sac 
edema and hemorrhaging (Henry et al., 1997; Spitsbergen et al., 1991). 

The exact mechanism underlying this pathology is not well understood, but appears 
to be, at least in part, due to the ability of these agents to cause vascular derangements, 
possibly via disruption of endothelial cell barrier function, or alternatively, as a result of 
CYPlAl induction with resulting oxidative damage (Guiney et al., 1997; Stegeman et al., 
1995; Toborek et al., 1995). The possibility that these environmental pollutants may also 
act through endocrine disruptive mechanisms is highlighted by the fact that functional 
estrogen receptors are required for the augmentation of basic Fibroblast Growth Factor 
(bFGF)-induced uterine angiogenesis in female mice (Johns et al., 1996). The effect 
appears to be an AhR-mediated process as seen by the fact that those compounds which do 
not bind the AhR, do not produce endothelial cell dysfunction (Toborek et al., 1995). In 
addition, Arnt " embryos die in utero as a consequence of abnormal yolk sac angiogenesis 
(Maltepe et al., 1997). Finally, CYPIA induction in endothelium in early development in a 
trout model appears to correlate with mortality and the development of edemas prior to 
death, a process which is consistent with involvement of the AhR (Guiney et al, 1997). 



CHAPTER 2 
MATERIALS AND METHODS 



Materials 
Chemicals and Bioreagents 

TCDD was obtained from Midwest Research Institute (Kansas City, MO) through 
the National Cancer Institute Chemical Carcinogen Reference Repository. Benzo(a)pyrene, 
aminopterin, insulin, transferrin, endothelial cell growth supplement and ampicillin were 
purchased from the Sigma Chemical Co. (St. Louis, MO). Ovine prolactin was obtained 
from the National Hormone and Pituitary Program (NHPP). ['^^I]-EGF and [^H]-methyl- 
thymidine were purchased from Amersham Life Sciences (ArUngton Heights, IL) and [a- 
^^PJdCTP and [a-'-P]UTP from ICN Biomedicals Inc. (Irvine, CA). The Prime It® II 
random primer labelling kit and Nuctrap® probe purification columns were purchased from 
Strategene (La loUa, CA) and ExpressHyb hybridization buffer from Clontech Laboratories 
Inc. (Palo Alto, CA). The CellTiter 96^^ nonisotopic cell proliferation assay kit was 
obtained from Promega (Madison, WI). Restriction and modifying enzymes were 
purchased from Promega (Madison, WI), New England Biolabs (Beverly, CA) or 
Gibco/BRL (Grand Island, NY). The Fisher Leukostat^M stain was from Fisher Scientific 
(Lexington, MA). p-Aminomethylbenzenesulfonamide agarose resin (pAMBS) was 
purchased from Sigma Chemical Co. (St. Louis, MO) and ProBond™ Resin from 
Invitrogen (San Diego, CA). Cell culture media, Lipofectin® and antibiotics were from Life 
Technologies (Gaithersburg, MD) and Sigma Chemical Co (St. Louis, MO), with the 
exception of fetal bovine serum which was obtained from Hyclone Laboratories (Logan 
UT). Plasminogen, thrombin and fibrinogen were purchased from CALBIOCHEM® 



16 



17 

Biochemicals (La JoUa, CA). Low gelling temperature Sea Plaque Agarose^M was obtained 
from FMC® BioProducts (Rockland, ME), enterokinase from Biozyme Laboratories 
International Ltd. (San Diego, CA), G418 sulphate from CELLGRO (Herndon, VA), and 
bovine dermal collagen and Matrigel from Collaborative Biomedical Products (Bedford, 
MA). All other chemicals were reagent or molecular biology grade and were obtained from 
standard commercial sources. 

Recombinant cDNA Clones. Vectors and Plasmids 

Plasmids containing cDNA for EOF receptor (pE7), TGF-a (phTGFl-10-3350), 
c-myc (pGl-5'-c-myc), CYPlAl (phPl-450-3'), uPA (pHUK-8), TIMP-1 (pALP-181- 
EPA57), TIMP-2 (pSS38), TNF-a (pAW739) and P-actin (HHCI89; 65128) were 
obtained from the American Tissue Type Culture Collection (ATCC) (Rockville, MD). The 
plasmids containing cDNA for CYPIBI (Sutter et al., 1994) and DL-lp (Sutter et al., 1991) 
were kindly provided by Dr. William Greenlee (University of Massachusetts, Worchester, 
MA). Full length cDNAs for PLP-B and PLP-C (Clone C-308) were generous gifts of Dr. 
Mary Duckworth (University of Manitoba, Canada) and Dr. Michael Soares (University of 
Kansas, Kansas City, KS), respectively. For Northern blot analyses, the probes used 
were a 1.0 kb EcoRL fragment for CYPlAl, a 2.4 kb CM fragment for EGF receptor, a 
1.6 kb Sad fragment for c-myc, a 1.5 kb Pstl fragment for uPA, a 410 bp AvaVHincU 
fragment for TIMP-1, a 790 bp EcoRJ/Xbal fragment for TIMP-2, a 1.5 kb Eagl fragment 
for CYPIBI, a 1.4 kb Eagl fragment for IL-lp, a 1.3 kb Hindlll fragment for TNFa and 
a 1.1 kb EcoRl fragment for P-Actin. The mammalian expression vector pMXSND (Lee 
and Nathans 1988) was generously provided by Dr. Daniel Linzer (Northwestern 
University, Evanston, IL). The bacterial expression vector pET22b(-l-) (Novagen, San 
Diego, CA) and all other non-expressing bacterial strains were provided by Dr. William 
Farmerie and G. Van Heeke (University of Florida, Interdisciplinary Center for 



18 

Biotechnology Research [ICBR], Protein Expression Core, Gainesville, FL). 
Oligonucleotide primers were synthesized by the ICBR DNA Synthesis Core. 

Antibodies and Antisera Generation 

A polyclonal sheep anti-human EGF receptor antiserum was acquired from Upstate 
Biotechnology Inc. (Lake Placid, NY) and the polyclonal goat anti-rat CYPl Al antiserum 
from Gentest (Woburn, MA). The horseradish peroxidase labeled goat anti-sheep IgG and 
rabbit anti-goat IgG were purchased from Bio-Rad Laboratories (Hercules, CA). Rabbit 
polyclonal antisera against PLP-B was previously generated in our laboratory (Ogilvie et 
al., 1990a). To generate antiserum against recombinant PLP-C, a New Zealand White 
rabbit was injected subcutaneously with 220 [ig of purified recombinant PLP-C in Freund's 
complete adjuvant, and three booster injections of 300 |j,g hCAII-PLP-C fusion in Freund's 
incomplete adjuvant were administered at 1 week intervals. Two weeks after the final 
booster, the animal was bled and hCAIE cross-reactivity was absorbed out during an 
overnight incubation with hCAII-pAMBS resin. The hCAH-pAMBS resin was generated 
by incubating hCAII with pAMBS overnight. 

Methods 

Cell Cultures and Chemical Treatments 

RL95-2 Endometrial Carcinoma Cells The human endometrial carcinoma cell hne RL95-2 
was obtained from ATCC and maintained in DMEM:HAM'S F-12 (1:1) supplemented with 
10% (w/v) FBS in a humidified atmosphere containing 5% CO? at 37°C. All media 
contained penicillin and streptomycin at 100 p.g/ml. Media was changed every 2-3 days, 
and all experiments initiated when cells were at approximately 50-75% confluence. Cells 
were cultured in the presence or absence of chemical treatments, added in either DMSO, 



19 

ethanol or buffered aqueous solution. Stock solutions of TCDD and B(a)P were prepared 
in DMSO and added to cultures with final DMSO concentrations at 0.1% (w/v). 
Appropriate vehicles were added to cultures as controls. 

Nb2 cells The Nb2-1 Ic subline was kindly provided by Dr. Paul Kelly (INSERM, Paris, 
France). It was maintained in RPMI 1640 media supplemented with 100 U/ml penicillin, 
100 |ig/ml streptomycin (GIBCOBRL, Grand Island, NY), 50 |.iM 2-mercaptoethanol, 
10% (w/v) horse serum (HS) and 10% (w/v) FBS. Starvation media was composed of the 
maintainence media minus FBS. 

Chinese Hamster Ovarv (CUO) Cells The CHO cell line was obtained from ATCC and 
routinely maintained in HAM'S F-12 supplemented with 100 U/ml penicillin, 100 f^g/ml 
streptomycin and 10% (w/v) FBS. For the collection of recombinant PLP-B, the stably 
transfected cells were transferred to HAM'S F-12 with 25-40 nM CdCl,, and conditioned 
media aspirated every 24-48 hr and stored at -20°C. 

Retinal EndotheUal Cells Human capillary endothelial cells were isolated from 

collagenase-digested donor retinas using the technique described by del Vecchio and 
Schaffer (1991) and modified as in Grant and Guay (1991). The purity of the primary 
endothelial cell cultures was evaluated by phase contrast microcopy and immunoflourescent 
labeling with acetylated low-density lipoprotein labeled with l,rdioctadycl-3,3,3',3' 
tetramethyl-indocarbocyanin perchlorate (Dil-Ac-LDL, Biomedical Technologies, 
Stoughton, MA). Cells from passage 5-7 were used in the migration stiidies. The primary 
endothelial cultures were grown on 0.2% (w/v) gelatin coated plates and maintained in 
DMEMwith 10% (w/v) FBS, 0.5 ^ig/ml insulin and tiansferrin, 100 p,g/ml streptomycin, 
100 U/ml penicillin, 0.5 |ig/ml Amphotericin B and 0.15 |ig/ml endothelial cell growth 
supplement. 



20 

Expression and Purification of the hCAII-PLP-C Fusion Protein 

Plasmid p0304/PLP-C23 was transformed into the bacterial expression host 
JM109(DE3) according to the procedure of Hanahan (1993). Cultures containing 
p0304/PLP-C23 were grown at 370C overnight in Luria Broth supplemented with 
ampicillin (50 !ig/ml). The overnight culture was diluted 1:500 in fresh media and grown 
at 370c until the optical density measured at 550 nm reached 0.6-0.8. Expression of the 
fusion protein was induced by adding 0.1 mM isopropyl (3-thiogalactosidase and 12.5 [xM 
ZnCl2. Following a 5 hr incubation at room temperature, cells were harvested by 
centrifugation at 1500 xg for 15 min and the cell pellet was stored at -80°C. 

Cells were lysed by freeze-thawing and sonication and resuspended in cold 50 mM 
Tris/0.5 mM EDTA (pH 7.8) containing O.lmM PMSF and 25 |iM ZnCl2. The suspension 
was incubated with DNasel for 1 hr and then centrifuged for 30 min at 15000 x g . The 
supernatant was adjusted to pH 8.7 and incubated with 3 ml pAMBS agarose resin 
overnight. pAMBS affinity chromatography utilizes hCAII as the purification ligand. The 
resin was recovered by centrifugation and washed with 30 bed volumes of 0. 1 M Tris/0.2 
M K2SO4/O.5 mM EDTA (pH 9.0) followed by the same buffer at pH 7.0 until the 
absorbance at 260 and 280 nm was effectively zero. The hCAII-PLP-C fusion protein was 
eluted from the resin with 0.4 M KSCN/0.1 M Tris/0.5 mM EDTA (pH 6.8), and 
concentrated using centriprep-10 concentrators (Amicon). The retentate was dialysed for 
3-4 days against 1000 volumes of 50 mM Tris/2 mM Ca2+ (pH 8). All purification steps 
were performed at 4°C. Protein concentration of all samples was determined by the method 

of Bradford (1976). 

The dialysed protein was digested (lU enterokinase:3 [ig protein) at 37''C for 30- 

36 hr, and the mixture was then incubated with the pAMBS agarose resin overnight. The 

resin was collected by cenUifugation and the supernatant containing recombinant PLP-C 

was concentrated, dialysed and stored at -20°C. 



21 



Construction of the pET22b(+)PLP-C Bacterial Expression System 

Expression Vector: The expression construct was generated by modification of the 
original hCAII-PLP-C fusion construct. The hCAII-PLP-C plasmid construct was double 
digested with Eael/HincE to generate a 751 bp fragment containing the PLP-C cDNA 
insert. The bacterial expression vector pET22b(+) was digested with Ndel/Notl. Both 
fragments were gel purified on 1% (w/v) agarose and the bands isolated with the Quiaex 
gel extraction kit (Quiagen, Chatsworth, CA). Insertion of the PLP-C DNA into the 
pET22b(+) system with a polyhistidine linker region required that oligonucleotide adaptors ! 

containing the polyhistidine sequence be annealed to the 5' end of the PLP-C insert. i 

Upper Primer: 46 mer 5'- TAT GGG CCA TCA TCA TCA TCA TCA TCA 
TCA TCA TCA CGT GAG CGG C -3'. Lower Primer: 44 mer 5'- GCC GCT Ij 

CAC GTG ATG ATG ATG ATG ATG ATG ATG ATG ATG GCC CA -3'. To faciUtate 
this procedure a three way ligation reaction was set up with the 750 bp PLP-C insert, 
pET22b(+) and the oligo adaptors in the presence of T4 DNA ligase overnight at 4°C. Five 
|j,l of the ligation reaction was used to transform competent JM109 cells and viable 
recombinant colonies were screened on LB-Amp plates for the presence of the PLP-C 
insert using a Ndel digestion. A single positive clone was transformed into the bacterial 
expression host BL21(DE3). 

Purification of Recombinant His-PLP-C: The recombinant bacterial expression system 
was cultured as previously described for the hCAII-PLP-C fusion protein and stored at 
-20°C until ready for His-PLP-C purification. The bacterial pellets were lysed in 20 mM 
Tris, 5 mM Imidazole, 0.5 M NaCl (pH 7.9) containing 0.1 mM PMSF. The suspension 
was incubated with DNasel for 1 hr, and then centrifuged for 30 min at 15000 x g. 
Recombinant His-PLP-C was isolated from the supernatant via the ability of the Histidine 
tag sequence on the protein to bind to immobilized divalent nickel using the His • Bind® 
metal chelation resin (Novagen, San Diego, CA). The supernatant was passed over the 



22 

nickel column, followed by 30 volumes of wash buffer (60 mM Imidazole, 0.5 mM NaCl, 
20 mM Tris pH 7.9). The bound protein was eluted in elution buffer (0.75 M Imidazole, 
0.5 M NaCl, 20 mM Tris pH 7.9) and collected in 1ml fractions which were analysed on a 
UVA^is spectrophtometer at 280 nm for the presence of protein. Protein fractions were 
dialysed into 50 mM Tris, 5 mM Ca^^ (pH 7.9). The dialysed protein was incubated with 
enterokinase at 37°C overnight, and centrifuged at 1500 x g for 10 min to remove any 
precipitate. The supernatant containing cleaved recombinant PLP-C was then stored at 
-20"C. 

Amino-terminal Sequence Analysis 

N-terminal amino acid sequence was determined by Edman degradation using a gas 
phase protein sequencer at the University of Florida Interdisciplinary Center for 
Biotechnology Research (ICBR) Protein Chemistry Core. 

Construction of the PLP-B Mammalian Expression System 

Expression Vector : The pGEM3-PLP-B clone (Duckworth et al., 1988) was doubly 
digested with EcoRi/HincIL in order to release the 700 bp cDNA insert containing the 
complete coding sequence for PLP-B. The digest was run on 1% (w/v) agarose gel and the 
700 bp band excised from the gel with a scalpel and purified using the Quiaex gel 
purification kit (Qiagen, Chatsworth, CA). pMXSND (Lee and Nathans, 1988) was 
digested with Xhol and gel purified. Both cDNA framents were blunt ended by incubation 
with 2 mM dNTP and 5 U of Klenow at 25°C for 20 min, then the enzyme was heat 
inactivated by incubation at 75°C for 15 min. pMXSND was further dephosphorylated by 
incubation with 40 U of Calf Intestinal Alkaline Phosphatase (CIAP) for 90 min at 37°C to 
inhibit self ligation. Ligation between PLP-B and dephosphorylated pMXSND was set up 
in a 20 |j.l volume with 4 U of T4 DNA Ligase at 4°C overnight. Ten |il of the ligation 



23 



mixture was used to transform competent JM109 cells. Viable colonies were used to 
generate minicultures in Luria Broth (LB) and plasmid DNA isolated with the Wizard 
Miniprep Kit (Promega, Madison, WI). Diagnostic digests were performed by digestion 
with BamHI to ascertain the presence and correct orientation of the PLP-B insert. Clone 1 1 
possessing the insert in the correct orientation was transformed into competent JM109 cells 
and a DNA maxiprep performed using a Qiagen Maxiprep kit (Qiagen, Chatsworth, CA) to 
produce transfection grade plasmid DNA. 

Transfection and Stable Mammalian Expression of PLP-B: The pMXSND-PLP-B 

construct was transfected into approximately 50-75% confluent CHOKl cells. Transfection 
grade plasmid DNA was diluted to 1 mg/ml in ddHjO. Five ml of Lipofectin® was added 
to 100 111 of OptiMEM for 45 min. pMXSND-PLP-B 1 \ig and 2 ^g were added to 100 |il 
of OptiMEM and then mixed with the Lipofectin®. The mixture was allowed to sit for 15 
min prior to addition of 1.8 ml of HAM'S F-12. The wells of the plates were washed with 
serum-free, antibiotic-free media followed by the addition of the Lipofectin-DNA media. 
I Plates were incubated at 37°C and 5% CO,. After 24 hr the media was aspirated and 4 ml 

of HAM'S F-12, 10% (w/v) FBS added. Cells in each well were trypsinized 48 hr later, 
diluted 1:100 into G418 selection media (HAM'S F-12, 10% (w/v) FBS supplemented 
with 800 [ig/xnl G418) and plated in 10 cm dishes. Colonies were picked using a sterile 
loop and placed in individual wells of a 96 well plate in selection media. After a week, 
seven wells were trypsinized and passaged into each of two 10 cm culture plates in 
selection media. After allowing the cells to attach for 2 days, the cells were fed with 
methotrexate selection media, (10 |i.M methotrexate, 10% (w/v) dialysed FBS, 500 M-g/ml 
G418, HAM'S F-12). Cultures were grown to 50-75% confluency and one of each of the 
two plates treated with 50 nM CdClj for 24 hr prior to RNA isolation, to confirm the 
presence of the message for PLP-B by Northern blot analysis using a ^-P-labelled PLP-B 
cDNA probe. Further selection was performed using methotrexate at concentrations up to 
180 |iM. Cells were washed in Hank's buffer and incubated in seaim-free HAM'S F-12 



24 



with 25 nM CdCl2 and conditioned media collected at 24-48 hr intervals and stored at - 
20°C. 

Basal Zone Explant Conditioned Media 

Timed pregnant Sprague Dawley rats were obtained from Holtzman (Madison, 
WI). Animals were housed in a temperature controlled room with 12 hr light/dark cycles 
and given food and water ad libitum. Animals were sacrificed on day 16-19 of gestation 
under sodium pentobarbital anesthesia. The uteri were removed, and placental basal 
(junctional) zones were isolated using forceps and dissecting scissors. Basal zone tissue 
was minced and incubated under sterile conditions in modified Eagle's medium (175 mg 
tissue/5 ml medium) for 24 hr at 37°C under 47.5% 02:2.5% CO2:50% N2. The culUire 
medium was supplemented with 3 mg/ml glucose, 100 U/ml penicillin, 100 |ig/ml 
streptomycin, 0.25 iig/ml amphotericin B, 0.2 U/ml insulin, 1% v/v non-essential amino 
acids, 1% v/v MEM vitamins, and 1.5 lag/ml methionine (1/10 normal concentration). 
After incubation the conditioned medium was centrifuged at 1000 x ^ for 10 min to remove 
debris and stored at -20°C. 

Immuno- Affinity Protein Purification: 

The immunoaffinity columns for PLP-B and PLP-C were generated by linking 
generated polyclonal antipeptide antisera (Ogilvie et al., 1990b) to an activated agarose 
support matrix Affi-GeF'^ (Bio-Rad, Hercules, CA) according to the manufacturer's 
instruction. For purification of native PLP-B and PLP-C, as well as recombinant PLP-B, 
conditioned media at neutral pH was passed over the column and the column washed with 
30 bed volumes of buffer (0.5 M NaCl, 10 mM phosphate, pH 7.0); bound protein was 
eluted in 0.2 M Glycine, pH 2.8 and neutralized with K2HPO4, pH 9.2. Neutralized native 
protein was concentrated using Amicon centriprep concentrators and stored at -20°C until 



25 



required. Protein concentrations were estimated by comparison with Coomassie stained 
BSA standards on SDS-PAGE minigels. 

Endothelial Cell Migration Assay 

Endothelial cell migration assays were performed essentially as in Grant et al (1987) 
using modified Boy den Chambers (Neuro Probe Inc., Cabin John, MD). Human retinal 
endothelial cells were trypsinized from a T75 flask, and pelleted by centrifugation at 500 x 
g for 5 min at room temperature. The cells were resuspended in Hank's buffer and washed 
twice. Cells numbers were evaluated on a hemocytometer after staining with 0.25% 
methylene blue solution. Approximately 7000 cells in 27 |il of serum-free growth media 
were aUquoted into the lower wells of the migration chamber. The wells were overlaid 
with a porous bovine dermal collagen coated membrane (5 iiim diameter pore size) and the 
gasket seal and upper chamber attached. The apparatus was inverted at 37°C and 5% CO2 
for 90 min to allow for cellular attachment, after which test protein solutions in serum-free 
media were added in a total of 50 p,l/well in sextuplicate. 10% FES and serum-free media 
were used as positive and negative controls, respectively. The apparatus was incubated 
overnight at 3TC/5% CO2 to facilitate migration of attached cells. The apparatus was 
disassembled, backside (cellular attachment side) scraped, and the membrane stained in 
Leukostat''^ stain. The membraue was then mounted on a glass slide for evaluation by 
counting the numbers of migrated cells/well under a light microscope. For migration 
assays, cells were evaluated in quadruplicate wells for each treatment regimen. 

In Vitro Invasion Assay 

The Matrigel invasion assay was performed using the modified Boy den Chamber 
apparatus as in the endothelial cell migration assay (Grant et al, 1987). RL95-2 cultures 



26 

were incubated with B(a)P and TCDD for 48 hr. Cells were trypsinized and pelleted by 
centrifugation at 500 x g for 5 min. The cells were resuspended in Hank's buffer and 
washed twice. Cells numbers were determined using a hemocytometer. Approximately 
7000 cells in 27 |i.l of complete media in the presence of the respective chemicals were 
aliquoted into the lower wells of the Boy den chamber. The wells were overlaid with a 
porous Matrigel-coated polyvinyl-pyrrolidone-free polycarbonate membrane (8 micron 
diameter pore size) and the gasket seal and upper chamber attached. The apparatus was 
inverted at 37°C/5% CO2 for 90 min to allow for cellular attachment, after which time 50 |i,l 
of complete media was added to the upper wells. The apparatus was incubated for 36 hr at 
37°C/5% CO2, at which time the apparatus was disassembled, the backside of the 
membrane (cellular attachment side) scraped, and the membrane stained in Leukostat^i^ 
stain. The membrane was then mounted on a glass slide for evaluation under a light 
microscope by counting the number of cells per well which had invaded the membrane. 
For these assays, cells were counted in quadruplicate wells for each treatment regimen. 

Nb2 Assay for Lactogenic Activity 

The Nb2 proliferation assay was performed essentially as described by Gout et al 
(1980). The lactogenic response was evaluated using [^H]-thymidine incorporation and 
MTT dye conversion as indicators of the growth response. Log phase Nb2 cells in 
suspension were collected by centrifugation, washed, and resuspended in starvation media 
overnight. Cells were again collected by centrifugation and 4 x lO'* cells in 100 |il were 
added to the triplicate wells on a 96 well plate. Ovine PRL (NIDDK) in concentrations 
between 0.1-100 ng/ml was added in triplicate wells in 50 |il aliquots as the standard 
stimulatory response, while native and recombinant PLP-B and PLP-C were added in the 
same volume. The cultures were harvested onto microfiber filter paper after a 4 hr [^H]- 
thymidine pulse using a Brandel cell harvester at 48 hr (Gaithesburg, MD), and [^H]- 



27 

thymidine uptake was measured using a Beckman LS 7000 liquid scintillation counter. 
Alternatively, Nb2 cells and the formazan product were solubilized at 48 hr after a 4 hr 
exposure to MTT, and the absorbance measured at 595 nm on an Elisa plate reader (Adler 
et al., 1994). 

EGFR Binding Assay 

RL95-2 cells were grown for 24-96 hr in monolayer culture in the presence of 0-50 
nM TCDD in serum-free medium or in the presence of 2% (w/v) or 10% (w/v) FBS 
supplemented DMEM/HAM'S F-12. Cells were washed three (3) times with cold PBS 
(pH7.4), scraped from plates with a rubber policeman and pelleted by centrifagation. Fifty 
|j.g of protein (whole cells) was incubated in serum-free DMEM-HAM'S F-12 containing 
0.1% (w/v) BSA, with 400 pg ["^I]-EGF (8 x 10^ cpm) in the presence or absence of 100 
ng unlabelled EGF for 16 hr at 4°C. Incubations were stopped and unbound radioactivity 
removed by washing cells twice in cold PBS. Total bound cpm was measured using a 
gamma counter. Specific binding was expressed as the difference between radioactivity 
bound in the absence (total) and presence (non-specific binding) of excess unlabelled EGF. 

Western Immunoblot Analysis 

General Procedure: Cells were rinsed two to three times, collected with a cell scraper, 
and lysed in 0.5-1 ml PBS using three freeze-thaw cycles. The membrane fraction was 
obtained after centrifugation at 10000 x g for 15 min at 4°C and then resuspended in 
ddHjO. Alternatively, cells were scraped into lysis buffer containing 10 mM Tris, 0.14 M 
NaCl, 0.5% (w/v) Na-deoxycholate, 1% (v/v) Triton-X 100, 1 mM phenyl-methyl 
sulfonyl flouride (PMSF), and 1 }xg/ml each of leupeptin and aprotinin. Cells were then 
incubated with the lysis buffer for 1 hr at 4°C on an orbital shaker and the supematant was 
recovered after centrifugation at 10000 x g for 15 min at 4°C. Samples of membrane or 
total cell lysate protein (50-100 |ig) were then separated by 7.5% or 10% SDS-PA gels and 



28 

transferred electrophoretically to nitrocellulose filters using 25 mM Tris, 192 mM glycine 
buffer at pH 8.2, with 20% (v/v) methanol according to the method of Towbin et al (1979). 
EGF Receptor and CYPlAl Protein: For EGF receptor and CYPlAl protein analysis, 
immunostaining was performed as previously described in Wang et al (1987). The 
nitrocellulose membranes were washed in 20 mM Tris containing 0.1% (w/v) Tween 20 
and 0.9% (w/v) NaCl, pH 7.5 (TTBS) for 15 min, and then 3% (w/v) gelatin for 30 min. 
The membrane was then sequentially incubated with polyclonal anti-human EGF receptor 
antiserum diluted to 1 [ig/25ml in TTBS without Tween 20 or preimmune sheep serum for 
2 hr, followed by horseradish peroxidase labeled goat anti-sheep IgG for 60 min. 
Alternatively, for CYPlAl the membrane was incubated with polyclonal goat anti-rat 
CYPlAl (1:1000 dilution) or preimmune goat serum for 2 hr, followed by horseradish 
peroxidase conjugated anti-goat IgG for 1 hr. Bands were visualized by incubation with 3- 
amino-9-ethylcarbazole in the presence of 0.015% (v/v) hydrogen peroxide. 
Lnmunoreactive bands were quantitated by scanning the nitrocellulose filters on a Microtek 
ScanMaker II scanner and NIH image software. 

PLP-B and PLP-C Protein: The blotted nitrocellulose membranes were blocked in 3% 
gelatin for 30 min. The membrane was then sequentially incubated with either polyclonal 
rabbit anti-rat PLP-B or PLP-C antiserum diluted 1:1000 in TBS or preimmune rabbit 
serum for 2 hr. This was followed by incubation with horseradish peroxidase labeled goat 
anti-rabbit IgG for 60 min. Immunoreactive bands were visualized by incubation with 3- 
amino-9-ethylcarbazole in the presence of 0.015% hydrogen peroxide. Immunoreactive 
bands were quantitated as described above. 

RNA Isolation & Northern Blot Analysis 

Total cellular RISfA was isolated from TCDD-treated cell cultures by acid guanidium 
thiocyanate phenol-chloroform extraction according to Xie and Rothblum (1991). For 
Northern blotting, 40 p.g of total cellular was denatured, fractionated on a 1.5% (w/v) 



29 

agarose formaldehyde gel, and transferred to nylon membranes. cDNA probes were 
labelled with [a-^'P]-dCTP using a random primer labelling kit. Prehybridization was 
carried out in commercially available ExpressHyb buffer at 68°C for 1 hr, and hybridization 
in the same buffer for 2-4 hr after addition of the ''^P-labelled probe. The membranes were 
washed three times in 2 X SSC, 0.1% (w/v) SDS at room temperature for 10 min, then 
twice in 0.1 X SSC, 0.1% (w/v) SDS at 60-65°C for 30 min. Transcripts were then 
visualized by autoradiography. Blots were later stripped and rehybridized with ^^P-labelled 
cDNA for p-actin as a loading and transfer control in order to facilitate quantitation by 
optical scanning. 

Nuclear Run Off Assay 

Nuclei were prepared by a detergent lysis protocol (Ausubel et al., 1987). Actively 
proliferating cultures of RL95-2 cells were exposed to 10 nM TCDD or 0.1% (v/v) DMSO 
for 40 hr, after which the cells were washed twice with cold PBS, scraped from the plates 
with a rubber policeman and pelleted by centrifugation at 500 x ^ for 5 min. The cell pellet 
was resuspended by vortexing in 4 ml of NP-40 lysis buffer [10 mM Tris, 10 mM NaCl, 3 
mM MgCh, 0.75% (v/v) NP-40 pH 7.4], placed on ice for 5 min, after which nuclei were 
collected following a second round of centrifugation at 500 x g. This process was repeated 
and nuclei resuspended in 500 \il of glycerol storage buffer [50 mM Tris, 5 mM MgClj, 
0.1 mM EDTA, 40% (v/v) glycerol, pH 8.3] per 10^ nuclei. Nuclei were stored at -80°C 
in 200 p-l aliquots until ready for use. 

Nuclear runoff assays were performed essentially according to Srivastava et al 
(1993) and Merscher et al (1993) with minor changes. Frozen nuclei (= 4 x 10^) per 
reaction were thawed and mixed with an equal volume of 2 X reaction buffer (10 mM Tris, 
5 mM MgCl2, 0.3 M KCl, 5 mM DTT, 0.2 mM EDTA, 4 mM ATP, GTP, CTP) and 75 
fiCi [a-^^P]UTP. The reaction was allowed to proceed in a room temperature water bath 
for 30 min at which time 50 U DNasel was added and the reaction allowed to continue for 



30 

5 min. After labelling, the nuclear suspension was mixed with 1.2 ml of guanidium 
isothiocyanate reagent (Xie and Rothblum, 1991), 130 i^l chloroform- isoamylalcohol 
(24:1), and vortexed. The reaction tube was left on ice for 10 min and the aqueous layer 
collected after centrifugation at 10000 x g for 10 min and incubated with an equal volume 
of isopropanol at -80°C overnight. The following day the RNA pellet was collected after 
centriftagation at 10000 x g for 10 min and the pellet washed once in 70% (v/v) ethanol. 
The pellet was air dried and dissolved in 0.5% SDS and 1 jil used to determine activity 
using a scintillation counter. Equal amounts of cpms («10^) from DMSO control and 
TCDD experiments were added to 2 ml of ExpressHyb buffer and hybridized with 
j appropriate cDNAs on nylon membranes at 65°C for 48 hr. Membranes were then washed 

in 2 X SSC at room temperature for 30 min, then by 0. 1 X SSC, 0. 1 % (w/v) SDS at 62''C 
for 1 hr. Dot blots were visuahzed by autoradiography following 2-5 day exposure at - 
80°C. X-ray films were scanned using a Microtek ScanMaker 11 scanner and spot 
intensities quantitated by densitometry with each message standardized against p-actin as 
control. 

Fibrin Zymography 

Fibrin zymography was performed according to the method of Granalli-Pipemo and 
Reich (1978) with modifications by Cheng et al (1991) and Nakamura et al (1995). 
Briefly, RL95-2 cells were seeded into 10 cm plates at 5 x 10^ cells per plate and allowed to 
grow for three days. Cultures were exposed to TCDD and B(a)P for 36 hi" and the cultures 
washed in Hank's buffer before the addition of equal volumes of serum-free media to each 
plate. Conditioned media was collected from control and treated cultures after 12 or 24 hr. 
Thirty |i,l of CM was mixed with SDS sample buffer and run under non-reducing 
conditions on a 10% SDS-PA gel. Gels were then inmiersed in 50 mM Tris, 5 mM CaClj, 
2.5% (v/v) Triton X-100 three times for 15 min to remove the SDS, and then twice more 
(50 mM Tris, 5 mM CaCl,, pH 8.1) for 15 min to remove the Triton X-100. To detect the 



31 

plasminogen activator activity, the poly acryl amide gel was overlaid onto a 1% (w/v) low 
temperature gelling agarose gel containing 75 ill of Img/ml of plasminogen, 30 |li1 of 1 
U/ml thrombin, and 10 ml of 5 mg/ml fibrinogen, in phosphate buffered saline (PBS). The 
gels were incubated overnight in a humidified atmosphere at room temperature. 
Plasminogen activator activity was detected as a lysed zone on the agarose gel after staining 
with Coomassie brilliant blue or amido black. Bands were scanned and fibrinolytic 
activities assessed by comparison of the areas of the lytic zones. 

Data Analysis 

All experiments were performed at least three times at concentrations and time 
points indicated unless otherwise stated. For analysis of scanned images, control lanes 
were standardized to 100% and all treatments assessed relative to controls for each indiviual 
experiment. For Northern blots loading was standardized to p-actin. One factor ANOVA 
analysis was performed to assess the dose-response effects with significance being 
determined at the p < 0.05 level. Numerical data averaged over several experiments was 
represented as Mean ± Standard Error of the Mean (SEM) and the Student's t-test used to 
analyse the data. Statistical analyses were performed using Microsoft Excel software 
program. 



CHAPTER 3 
EFFECTS OF TCDD AND B(a)P ON CELLULAR PROLIFERATION AND EOF 
RECEPTOR EXPRESSION IN THE RL95-2 CELL LINE 



Introduction 

Endometriosis is the ectopic growth of endometrial tissue outside of the uterus and 
has been described as a disorder which has characteristics of both a benign disease and an 
invasive neoplasm (Arnold et al., 1996). It is first and foremost a proliferative disorder in 
which endometriotic tissue is mislocated to and invades extrauterine sites. In view of 
recent evidence implicating environmental agents hke TCDD and cigarette smoking in the 
etiology of endometriosis (Cummings et al., 1996; Mattorras et al., 1995; Mayani et al., 
1997; Rier et al., 1993), our laboratory undertook an investigation as to whether or not 
these environmental agents are able to directly alter the cellular proliferation of endometrial 
cells. 

Human uterine endometrium proliferates in response to estrogen during the course 
of the female menstrual cycle. The growth and maintenance of endometriotic tissue is 
known to be dependent upon estrogen as evidenced by its extreme rarity in premenarchal 
giris and the association with exogenous estrogen administration in postmenopausal 
women (Goodman et al., 1989). Recent evidence suggests that the action of estrogen on 
the endometrium is mediated by EOF, with EOF being able to mimic the stimulatory effects 
of estrogen on DNA synthesis and lactoferrin gene expression in ovariectomized mice 
(Nelson et al., 1991). Estrogen also increases EOF binding and mRNA levels for the EOF 
receptor in uterine tissue (Lingham et al., 1988; Mukku and Stancel, 1985). 



32 



33 

TCDD and B(a)P have been shown to generally downregulate EGF receptor 
expression in different tissues and cell lines (Astroff et al., 1990; Guy da et al., 1990; 
Hudson et al., 1986; Sewall et al., 1993; Zhang et al., 1995), although upregulation of 
EGF expression by TCDD has been demonstrated developmentally (Abbott and Birnbaum, 
1990; Abbott et al., 1992). EGF receptor expression has been demonstrated in human 
endometriotic tissue (Huang and Yeh, 1994; Prentice et al., 1992), as well as in 
endometriotic lesions from surgically-induced animal models (Simms et al., 1991; Zhang et 
al., 1993a). Therefore, EGF receptor expression may have a role to play in the 
pathogenesis of endometriosis. 

Our study evaluated the potential changes in EGF binding and EGF receptor protein 
expression in endometrial cells subsequent to TCDD and B(a)P exposure. The goal of the 
study was to ascertain whether or not a potential correlation could be made between EGF 
receptor expression and cellular proliferation, as well as with the induction of the metabolic 
enzymes CYPlAl which is classically induced by TCDD and B(a)P exposure (Sutter et al., 
1994; Whitlock, 1989). 

Results 

Effects of TCDD and BfalP on CYPlAl and CYPIBI mRNA in RL95-2 Cells 

We initially examined the ability of TCDD and B(a)P to induce the expression of 
mRNA for CYPlAl, a classic functional biomarker of exposure to AhR agonists 
(Whitlock, 1989). Northern blot analysis of 0.1% (v/v) DMSO (control) treated RL95-2 
cells indicated that the 3.0 kb mRNA transcript for CYPlAl was virtually undetectable. 
However, 48 hr exposure to TCDD at a concentration of 0.1 nM TCDD was able to 
significantly induce CYPlAl mRNA expression with induction being maximal at the 1 nM 
TCDD level (Figure 3-lA). In data not shown, induction of CYPlAl mRNA by 10 nM 
TCDD was observed as early as 6 hr after exposure. Similarly, 10 [xM B(a)P exposure for 



34 

12 hr resulted in significant CYPl Al mRNA induction, with levels being maximal by 24 hi" 
and maintained for the 48 hr assay period (Figure 3- IB). The expression of CYPIBI 
mRNA, a second drug metabolizing enzyme recently shown to be induced by TCDD 
(Sutter et al., 1994), was further analyzed by Northern blot analysis. As shown in Figure 
3-lA, RL95-2 cultures express a constituitively low level of the 5.1 kb CYPIBI mRNA 
transcript which is readily induced by TCDD concentrations as low as 0. 1 nM. Hence the 
RL95-2 cell line appears to possess a functional AhR signal transduction system as 
described in Chapter one. 

Effects of TCDD on '"l-EGF Binding in RL95-2 Cells 

The ability of TCDD to alter the total specific binding of '"^I-EGF to intact RL95-2 
cultures was evaluated. Cultures treated with 1-100 nM TCDD for up to 96 hr did not 
exhibit any significant change in the total binding of '^"^I-EGF as compared to 0.1% (v/v) 
DMSO-treated controls (Figure 3-2). Total specific '^-^I-EGF binding was in the range of 
10-12% as has been previously demonstrated for this cell line (Korc et al., 1986). In this 
regard, it is pertinent to note that these experiments were performed at 4°C so as to 
minimize internalization of the transmembrane EGF receptor. 

Effects of TCDD and B(a)P on EGFR and CYPlAl Protein in RL95-2 Cells 

Exposure of RL95-2 cultures to 1 nM and 10 nM TCDD showed a marked 
induction of the 55 kDa CYPlAl protein band as determined by Western immunoblot 
analysis (Figure 3-3A). In data not shown, this induction of immunoreactive protein was 
shown to be concentration-dependent being maximal at 1 nM TCDD. Similarly, exposure 
to both 1 |iM and 20 )jM B(a)P also resulted in a dose-dependent induction of CYPlAl 
immunoreactive protein, although the level of induction was not as high as that exhibited by 
TCDD treatment (Figure 3 -3 A). The induction of immunoreactive protein correlated with 
the observed induction of mRNA transcripts for CYPlAl after exposure of RL95-2 



35 

cultures to both agents (Figure 3-1 A & B). The levels of the 170 kDa immunoreactive 
EGF receptor membrane protein band were unchanged after TCDD exposure at 
concentrations of 1 nM and 10 nM relative to 0.1% (v/v) DMSO controls (Figure 3-3A & 
B). In data not shown, no effect of TCDD was noted for exposures up to 96 hr. By 
contrast, 20 |i.M B(a)P almost totally eliminated the expression of EGFR immunoreactive 
membrane protein after a 48 hr exposure period (Figure 3-3A & B). This decrease in 
EGFR immunoreactive protein levels is an effect associated with B(a)P which has been 
previously observed in placental choriocarcinoma cells (Zhang et al., 1995). Thus the 
decrease in EGFR immunoreactive protein with B(a)P exposure was correlated with a 
corresponding induction in CYPl Al protein in this cell line. In contrast, TCDD treatment 
exhibited no obsei-vable decreases in EGFR protein levels while significantly greater 
CYPlAl induction was observed. 

Effects of TCDD and BfalP on Cellular Prohferation in RL95-2 Cells 

The effects of TCDD and B(a)P on RL95-2 cell proliferation were evaluated by the 
direct counting of viable cells. Under serum-free conditions, 10 |jM B(a)P significantly 
decreased the rate og growth of RL95-2 cells by 48 hr after exposure (Figure 3-4). 
Similarly, for cultures growing in medium containing 10% (v/v) FBS, the number of cells 
in 10 |iM B(a)P-treated cultures was lower than those of 0.1% (v/v) DMSO controls by as 
early as 24 hr (Figure 3-5). Furthermore, under serum-free conditions the number of 
viable cells in B(a)P treated cultures appeared to plateau after 48 hr with no further increase 
in cell numbers being observed for the remaining 24 hr of the assay. In contrast, 10 nM 
TCDD did not result in any significant change in cell numbers compared to control cultures 
for the duration of the 72 hr assay period. Thus 10 [iM B(a)P, but not 10 nM TCDD, 
adversely affected cellular proliferation of RL95-2 cells under both serum-free and 
complete media conditions. 



36 



Effect of TCDD and B(a)P on Steady State r-m.vc mRNA T^evels 

Northern blot analysis was next used to determine whether TCDD and B(a)P 
exposure of RL95-2 cultures could lead to alterations in the expression of the steady state 
levels of c^myc mRNA, a proto-oncogene associated with cellular proliferation (Vasti-ik et 
al., 1994). Data in Figure 3-6 show the presence of a strong constitutive level of 
expression for the 2.7 kb mRNA transcript in control cells. Expression was not 
significantly altered by up to 48 hr exposure to either 10 nM TCDD or 10 ^M B(a)P 
(Figure 3-6A & B). Hence the decreased abihty of RL95-2 cells to proliferate in the 
presence of B(a)P exposure is not correlated with a decreased level of c-myc mRNA 
expression. 

Discussion 

We chose to investigate the potential alterations in EOF receptor expression and 
cellular proliferation after treatment with TCDD and B(a)P in order to gain a better 
understanding of the effect these environmental agents might have on uterine growth and 
uterine disease pathologies. Endometriosis is an estrogen-dependent disease (Barbieri, 
1990) being rare before puberty or after menopause. Recent data indicate that estrogen 
actions on the uterus may be mediated through growth factors like EOF as well as increases 
in EOF receptor expression (Lingham et al., 1988; McBean et al., 1997; Nelson et al., 
1991). EGF has also been shown to be mitogenic in human endometriotic stromal tissue 
(Mellor and Thomas, 1994), while experiments with rat endometriosis models suggest that 
rat endometrial implants produce EGF and contain receptors for EGF (Simms et al., 1991). 
Furthermore, Danazol or gonadotropin releasing hormone analogues (GnRHa), chnical 
therapies for relieving the symptoms of endometriosis, significantly decrease the levels of 
immunohistochemical staining for the EGF receptor (Melega et al., 1991). 



37 

The RL95-2 cell line was found to express high levels of the 170 kDa 
immunoreactive EGF receptor protein in agreement with previous reports (Korc et al., 
1986, 1987; Lelle et al, 1993). In data not shown, these cells also exhibited the presence 
of the 5.6 kb mRNA transcript for the EGF receptor as has been demonstrated in other 
systems (Lin et al., 1991; Zhang et al., 1995). The specific binding of '"^I-EGF was not 
altered by exposure to concentrations of TCDD up to 100 nM for the duration of the 96 hr 
assay period compared to vehicle treated controls. This result was supported by Western 
immunoblot analysis for EGF receptor, while the levels of the 5.6 kb mRNA transcript for 
EGF receptor were also not significantly altered by TCDD treatment (data not shown). In 
contrast to TCDD, exposure of RL95-2 cultures to B(a)P resulted in a concentration-related 
decrease in the levels of the 170 kDa immunoreactive EGF receptor protein as determined 
by Western analysis. 

Tissue specific effects of TCDD and B(a)P on EGF receptor expression and 
j binding has been a well characterized toxicological observation (Pohjanvirta and Tuomisto, 

i 

I 1994). TCDD has been demonstrated to decrease the binding capacity of EGF receptors in 

the liver (Sewall et al, 1995), uterine tissue (Astroff et al., 1990) and in hepatoma cells 
and keratinocytes (Hudson et al., 1985, 1986; Karenlampi et al., 1983) and, in some 

.'ij cases, without a consequent decrease in steady state EGF receptor mRNA levels (Lin et al., 

1991). It should be noted that TCDD has been found to increase EGF receptor expression 
during early development in the embryonic mouse palate and ureteral epithelium (Abbott 
and Birnbaum, 1990; Abbott et al., 1992). In contrast, B(a)P has exhibited a similar effect 
j in downregulating EGF receptor protein levels in human keratinocyte and placental cell 

lines (Guyda et al., 1990; Hudson et al., 1985; Zhang et al., 1995). 

The mechanism by which these compounds are able to alter EGF receptor binding 
and expression are not well understood. Evidence that the effects of TCDD on the hepatic 
EGF receptor may be mediated via the Ah locus was found in congenic strains of mice 
differing at this locus (Lin et al, 1991). In this regard, prolonged exposure in rats 



38 

demonstrated that the EDjq for the decrease in receptor capacity was close to the EDjq for 
CYPlAl induction, which is known to be regulated transcriptionally by TCDD (Sewall et 
al., 1993). Furthermore, the inhibition of EGF-specific binding has been shown to be 
stereospecific in that a TCDD analogue unable to bind to the Ah receptor was not able to 
decrease EGF binding (Hudson et al., 1985). The results of our study indicate, however, 
that both TCDD and B(a)P were able to induce the expression of both CYPlAl mRNA and 
immunoreactive protein levels in the RL95-2 ceU line, while only B(a)P was able to 
selectively decrease EGF receptor expression. 

Karenlampi et al (1983) have proposed that electrophilic metabolites of polycyclic 
aromatic hydrocarbons may be responsible for the inhibition of EGF binding observed in 
mouse hepatoma cells. In this regard, TCDD is not readily metabolized and has a 
significant 7-10 year half-life in humans, while B(a)P is readily metabolized to a series of 
reactive species including epoxides, phenols and quinones (Gelbion, 1980). Thus the 
observed down-modulation of EGF receptor protein by B(a)P may be a direct effect of 
reactive metabolites which would not be observed with TCDD due to its resistance to 
metabolism. Altematively, EGF receptor down-modulation can be effected by changes in 
cytokines such as IL-1 and TNF (Bird and Saklatvala, 1990). As will be seen in Chapter 
Five, however, TCDD is able to induce the expression of these cytokines in RL95-2 cells 
without the observed decrease in EGF receptor protein. 

Uterine endometrium is highly responsive to estrogen action. Evidence that the in 
vivo administration of estrogen was able to stimulate c-myc expression at the transcriptional 
level in rat uterus has led to the suggestion that c-myc expression is related to estrogen- 
induced uterine cell proliferation (Murphy et al., 1987). Similarly, exposure of RL95-2 
cell cultures to estrogen has been demonstrated to induce the expression of c-myc in a time- 
dependent manner (Liu and Teng, 1994). In general, exposure of cells to estrogen in vitro 
has not been demonstrated to be mitogenic in isolated uterine cell cultures (Tomooka et al., 
1986), nor to induce c-myc mRNA in primary endometrial epithelial cells (Jouvenot et al.. 



39 

1990). Withdrawal of cells from the cell cycle by removal of growth factors leads to 
down-regulation of c-myc expression and may be a requirement for growth arrest (Waters 
et al., 1991). Furthermore, deregulated c-myc expression can induce apoptosis in 
fibroblasts deprived of growth factors (Evan et al., 1992), suggesting that this protein may 
serve to integrate the effects of different signaling pathways for cellular growth. 

Endometriosis is characterized by the extrauterine proliferation of endometrial 
tissue. Characterization of the effects of TCDD and B(a)P on proliferation of endometrial 
cell cultures would provide a better understanding of the potential contribution of these 
xenobiotics to the etiology of uterine disease. The present study indicates that B(a)P, but 
not TCDD, was able to significantly decrease the proliferative ability of RL95-2 cells in 
culture under both serum-free and complete growth media conditions. In fact B(a)P 
treatment resulted in virtual arrest of the growth of RL95-2 cells as seen in the plateau in 
cell numbers after B(a)P administration. In contrast, neither TCDD nor B(a)P were able to 
alter the expression of the steady state levels of mRNA for c-myc. Therefore the inhibition 
of cellular growth mediated by B(a)P does not appear to have a direct causal relationship 
with c-myc mRNA levels in this cell line. 

In summary, B(a)P, but not TCDD, is able to significantly decrease the expression 
of immunoreactive EGF receptor protein in RL95-2 cell cultures, and there does not appear 
to be a direct relationship with the ability of these agents to induce the expression of 
CYPlAl and IBl, members of the cytochrome P450 family. It is possible that reactive 
metabolites of B(a)P have a role to play in the process of EGF receptor down-modulation. 
These data correlate with the etiological association of TCDD with enhanced and B(a)P 
with decreased incidences of endometriotic lesions, respectively, insofar as previously 
published data has shown no significant differences in EGF receptor expression between 
normal and ectopic endometrium (Huang and Yeh, 1994; Prentice et al., 1992). 
Furthermore, B(a)P, but not TCDD, is able to inhibit the cellular prohferation of RL95-2 
cultures without a concomitant change in the expression of c-myc mRNA levels. Since c- 



40 

myc expression may not be directly related to the proliferative phenotype, but indirectly 
involved in the process, a direct correlation may not be observable with c-myc mRNA 
levels. 



41 



(A) 



TCDD(nM) 0.1 1 10 50 
CYPlAl 

CYPIBI 




P-ACTIN 



(B) 



B(a)P (10 ^iM) 



12h 



24 h 



48h 



CYPIAl 



P-ACTIN 




Figure 3- 1 . Northern blot analysis of the effect of TCDD and B(a)P on the 
CYPl Aland CYPIBI expression in RL95-2 cells. Total RNA was 
denatured, blotted, and hybridized with 32P-iabeled cDNA probes for 
CYPIAl and CYPIBI. (A) Dose-dependent induction of CYPl A 1 and 
CYPIBI by TCDD upon a 48 hr exposure to TCDD. (B) Time-dependent 
induction of CYPIAl mRNA after exposure to 10 |xM B(a)P. 



42 



15-, 



10 



o 
H 



5- 







\ 



■ T 



Hi 

J2 



. \ 



I 



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.".■, f y 

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'-■. \ 
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T 



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\ \ 

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y • 
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y 
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y 
\ \ 

y 
\ \ 

y 
\ \ 

y 
\ \ 



T 



.*-■ ^ 



ill Control 

133 1 nM TCDD 

10 nM TCDD 

\B 100 nM TCDD 



24 



48 



72 



96 



Time (hr) 



Figure 3-2. Graphical representation of the specific binding of [^^Sjj.gQp ^^ 
RL95-2 cells after treatment with 0-100 nM TCDD. Cells were incubated with 
TCDD in the presence of 10% FBS over a 96 hr period. Cellular protein, 50 ^g 
was incubated with 400pg [usjj.egf in the presence and absence of excess 
(100 ng) unlabeled EOF. Data represent the mean ± SEM of at least three 
separate determinations . 



43 



(A) 



TCDD 



Control 



InM 



lOnM 



BaP 

l^M 20|iM 



EGFR 

170kD ^ 



CYPlAl 
55kD 




(B) 



150 -1 



100 - 



50 - 



T 



I 



'mm 



Untreated 



InM 10nM 1 jiM 20nM 

TCDD BaP 



Figure 3-3. Effect of TCDD and B(a)P on EGFR immunoreactive protein levels 
in RL95-2 treated cultures. Actively growing cultures were treated with TCDD 
or B(a)P for 48 hr and total cell lysate run on 7.5% SDS-polyacrylamide gels and 
transferred onto nitrocellulose and probed with sheep anti-human EOF receptor 
antiserum. (A) Western blots of representative experiments. (B) Quantitation of 
changes in the level of EOF receptor protein in cultures treated with TCDD and 
B(a)P. Data are the mean + SE for three experiments. * P < 0.001 compared to 
controls. 



44 



KM! 

^^ 
o 

« 

© 

;z; 



400. 



300- 



200- 



>^ 



CONTROL 



E3 TCDD (10 nM) 



B(a)P (10 \M.) 



100- _ -r 

W^^^^M^ y y y 
l^^^^B \ V. \ 
I^^^^Hy y y y 
I^^^^V \ \ \ 
I^^^^^Hx ^ >- ^ 
^^^^^M \ \ \ 
^^^^^w* y y y 
^^^^^^H \ V. \ ' 
^^^^^K' y X y 

I 1^^^^ Lv^■VV^■V^ 



24 







T 






72 



TIME (hr) 



Figure 3-4. Quantitation of the effect of TCDD and B(a)P exposure on the 
proliferation of cultures of RL95-2 cells in serum-free media. Actively 
growing cultures were put into serum-free media in the presence of 0.1% 
(v/v) DMSO, 10 nM TCDD, or 10 ^M B(a)P as described in Materials and 
Methods. After designated times points cells were trypsinized and counted 
under a light microscope. Points represent mean ± SEM from three separate 
experiments. * P < 0.01 compared to control. 



45 



30 



O 



© 



10- 



0. 



CONTROL 



El TCDD (10 nM) 



B(a)P (10 mM) 







24 







- - - ■- »- » 




TIME (hr) 



Figure 3-5. Quantitation of the effect of TCDD and B(a)P exposure on the 
proUferation of cultures of RL95-2 cells in complete media. Actively growing 
cultures were put into complete media in the presence of 0.1 % DMSO, 10 nM 
TCDD, or 10 |iM B(a)P as described in Materials and Methods. After 
designated times points cells were trypsinized and counted under a light 
microscope. Points represent mean + SEM from three separate experiments. 
* P < 0.01 compared to control. 



46 



(A) 



B(a)P (10 ^iM) 
c-myc 



12h 



24h 



48h 




p-Actin 




(B) 



TCDD (10 nM) 



6h 



12 h 



24 h 



48 h 



C-myc 






P-ACTIN I 



wmmmmm mm^ 



Figure 3-6. Representative Northern blot analysis of c-myc mRNA. Cells were 
treated with TCDD or B(a)P up to a 48 hr time point and total RNA was isolated, 
denatured, blotted and hybridized with -^^P-labeled cDNA probe for c-myc as 
described in Materials and Methods. Representative Northern blot for (A) 10 |j.M 
B(a)P exposure and (B)10 nM TCDD treatment of RL95-2 cultures. 



CHAPTER 4 

EFFECTS OF TCDD AND B(a)P ON CELLULAR INVASIVENESS AND THE 

EXPRESSION OF uPA AND TIMPs IN A HUMAN ENDOMETRIAL CELL LINE 

RL95-2 



Introduction 

In endometriosis, uterine endometrial tissue is thought to mislocate to and invade 
extrauterine sites as a result of retrograde menstruation (Sampson, 1927). The growth 
regulation of endometriotic tissue, however, is poorly understood since a majority of 
women of reproductive age women exhibit retrograde flow, yet only around 10% of them 
will manifest symptoms of the disease, (Olive and Schwartz, 1993). This suggests that 
those women who develop endometriosis have endometriotic tissue which is more prone 
to implant and invade the peritoneum, possibly through the action of directed and localized 
extracellular proteolysis, which may involve plasminogen activators (PA) (Fernandez- 
Shaw et al., 1995). Alternatively, endometriosis is also associated with the presence of 
adhesions in the peritoneal cavity which has led to the suggestion that patients with 
endometriosis might have a deficiency in PA activity in the peritoneal fluid, resulting in 
more permanent adhesions because of reduced fibrin clearance (Malick, 1982). 

The process of cellular implantation is highly regulated because dysregulation may 
have profoundly undesirable consequences, as manifested in tumor metastasis. Figure 4-1 
presents a simplified schematic of potential actors in the process of peritoneal implantation 
based on the Sampson hypothesis (Edwards et al 1996; Stetler-Stevenson et al 1993). 
Urokinase PA (uPA), a serine protease, may play a role in cellular invasion by directly 
acting to degrade extra-cellular matrix (ECM) proteins such as fibronectin (Quigley et al., 
1987). In addition, uPA can also act through its ability to cleave plasminogen to plasmin, 

47 



48 

a protease which is capable of cleaving other ECM proteins as well as activating members 
of the family of enzymes known as matrix metalloproteinases (MMPs) (see Mayer 1990; 
Mignatti and Rifkin, 1993 for reviews). 

MMPs are a family of enzymes involved in ECM turnover and tissue remodeling 
(Stetler-Stevenson et al., 1993). Their activity can in turn be modulated by interaction 
with specific proteinase inhibitors known as tissue inhibitors of metalloproteinases 
(TIMPs), of which four have now been characterized (Boone et al., 1990; Docherty et al., 
1985; Greene et al., 1996; Hammani et al., 1996; Silbiger et al., 1994; Stetler-Stevenson 
et al., 1990). In the case of MMPs, active enzymes are inhibited by interaction with 
TIMPs which form tight-binding 1:1 complexes with the MMP enzyme active sites 
(Stetler-Stevenson et al., 1993). An imbalance between proteinases and their activators 
and inhibitors can be imphcated in a number of pathological states including tumor 
invasion (Liotta and Stetler-Stevenson 1991). 

The goal of this study was to determine whether TCDD and B(a)P could alter the 
invasive properties of endometrial cells as a potential etiological factor in uterine disease. 
TCDD has recently been demonstrated to induce the expression of uPA in human 
keratinocyte cell lines (Gaido and Maness, 1994, 1995). Our study thus evaluated the 
effect of TCDD and B(a)P on the ability of RL95-2 cells to invade matrigel membranes. 
The effect of these xenobiotics on the levels of uPA mRNA was also determined along 
with measurement of potential changes in the fibrinolytic activity of endometrial cells 
exposed to these agents. Finally, changes in the mRNA levels for TIMP 1 and 2 were also 
examined in order to determine whether these xenobiotic agents are able to up- or down- 
modulate other components of the tissue remodeling network. 



49 

Results 

Evaluation of the Effect of TCDD and Bfa)P on RL95-2 Cellular Attachment and 
Invasiveness 

The ability of endometrial cells to invade tissue at extrauterine sites is one of the 
requirements of the Sampson hypothesis (Sampson 1927). The ability of RL95-2 
endometrial cultures to invade matrigel membranes after TCDD and B(a)P exposure was 
evaluated utilizing a modified Boy den Chamber apparatus (Figure 4-2). The results are 
represented graphically in Figure 4-3 and indicate that 10 |iM B(a)P, but not 10 nM 
TCDD, was able to significantly inhibit the invasion of matrigel membranes as compared 
to control cultures (p < 0.001). The numbers of invasive cells was decreased in 
comparison to the control by up to 95% following a 48 hr exposure of cultures to 10 |xM 
B(a)P (Figure 4-3). Cellular invasion is a multi-faceted process involving cellular 
attachment and chemotaxis, as well as the degradation of basement membrane 
components. 

In order to ascertain whether or not the observed alteration in cellular invasiveness 
of B(a)P treated RL95-2 cultures was associated with cellular attachment, cells were 
allowed to attach to matrigel membranes for up to 24 hr. The numbers of attached cells 
were evaluated using a light microscope after nuclear staining. The results of a 2 hr 
attachment protocol are shown in Figure 4-4 A & B. Forty-eight hr exposure to 10 ^M 
B(a)P resulted in a significantly decreased ability of RL95-2 cells to attach to a matrigel 
membrane compared to DMSO treated controls (p < 0.001). This result was reproducible 
even for attachment periods of up to 24 hr (data not shown). It should be noted that the 
cells used in the attachment and invasion assays exhibited greater than 95% viability as 
determined by tryphan blue dye exclusion. 



50 

Effect of TCDD andBfalP on uPA mRNA Steady State Levels and Plasminogen Activity 
in RL95-2 Cells 

Northern blot analysis of the 2.8 kb mRNA transcript for uPA in RL95-2 cultures 
indicated that a high level of constitutive expression exists in control cells. Exposure of 
cultures to 10 nM TCDD, but not 10 ^M B(a)P, resulted in a 2-fold, time-dependent 
induction of uPA mRNA steady state levels (Figure 4-5A). The observed increase in 
steady state message for uPA mRNA was significant by 36 hr and remained elevated for 
the duration of the 48 hr assay period (Figure 4-6B). Furthermore, this increase was also 
dose-dependent, with significant induction not being observed below the 10 nM TCDD 
exposure level (Figure 4-6A & B). In contrast, neither 1 |a,M nor 10 |iM B(a)P exhibited 
any significant alteration in uPA mRNA levels after a 48 hr exposure of RL95-2 cells 
(Figure 4-6B). 

We next chose to investigate whether the level of fibrinolytic activity in the 
conditioned media from cultures exposed to TCDD and B(a)P was subsequently altered. 
Fibrin zymography is a classic assay for biological activity of uPA (Figure 4-7 A) which 
relies upon the ability of PA within the lanes on an SDS-PA gel to act upon plasminogen to 
generate active plasmin and hence produce fibrin degradation products which show up as 
lytic zones on Coomassie or amido-black stained gels (Granelli-Piperno and Reich, 1978). 
Analysis of the conditioned media from TCDD and B(a)P treated cultures, showed the 
presence of a single 54 kDa lytic band after staining. Evaluation of the relative sizes of the 
lytic bands generated by the conditioned media from treated cultures was performed by 
densitometric scanning. The analysis indicated that neither 10-100 nM TCDD, nor 10 |iM 
B(a)P significantly altered fibrinolytic activity in conditioned media from cultures of RL95- 
2 cells compared to 0.1% (v/v) DMSO controls (Figure 4-7B). 



51 

Effects of TCDD and BCalP on TIMP mRNA Expression in RL95-2 Cells 

We next evaluated the steady state mRNA levels for the TIMPs because of their 
role in the regulation of proteases important in the degradation of matrix membrane 
proteins. Northern blot analysis was performed after 48 hr exposure of RL95-2 cultures 
to 10 nM TCDD and 10 |jM B(a)P. Both TCDD and B(a)P treatments significantly 
increased the steady state levels of the 1 kb mRNA transcript for TIMP-l relative to DMSO 
treated controls (p < 0.05); however, B(a)P was able to induce relatively higher steady 
state levels of TIMP-l than was TCDD (Figure 4-8 A & B). In contrast, a time course 
analysis of TIMP-2 mRNA levels after treatment of cultures with 10 nM TCDD did not 
demonstrate any significant change in the levels of either the 1.2 or 3.5 kb mRNA 
transcripts (Figure 4-9). Thus there appears to be a selective increase in the expression of 
steady state levels of TIMP-l mRNA as opposed to TIMP-2 by TCDD and B(a)P 
exposure in the RL95-2 cell line. 

Discussion 

Rier et al (1993) demonstrated that the incidence and severity of endometriotic 
lesions in female rhesus monkeys was associated with their dietary exposure to TCDD in a 
dose-related manner. Study of a surgically-induced model of endometriosis in rodents 
similarly showed that the administration of TCDD and the pesticide methoxychlor (MTX) 
significantly promoted the growth of endometriotic sites (Cummings and Metcalfe, 1995; 
Cummings et al, 1996). In this regard, the physiological and molecular mechanisms 
whereby endometriotic tissue develops and persists outside of the uterine cavity and 
musculature are not well understood. On the basis of observed alterations in cellular and 
humoral immune function in endometriosis patients, it has been hypothesized that 
endometriosis may be the result of a decreased immune surveillance, recognition and 
destruction of misplaced endometrial tissue (Dmowski et al., 1994). Results of our 



52 

investigation into the potential alteration in immunological components by TCDD will be 
examined in Chapter five. The present chapter examined the data which relates to the 
potential role of TCDD and B(a)P in the alteration of factors involved in endometiial cell 
invasion and their potential contiibution to the promotion of endometriotic lesions. 

The data from the present study as presented in Figure 4-3 demonstrate that B(a)P 
produced a significant decrease in the ceU invasive activity of the RL95-2 endometrial cell 
line. In contrast, TCDD exposure did not significantly alter the ability of the RL95-2 cells 
to invade matrigel membranes. Our data with B(a)P is consistent with epidemiological 
findings that cigarette smoking is correlated with significantly reduced incidence of the i 

disease (Cramer et al., 1986; Mattorras et al., 1995). The fact that TCDD did not 

significantiy alter the cell invasive activity of the exposed cultures impUes that this agent [ 

I 
may not act to promote endometriosis by enhancing the implantation of uterine cells, but ; 

may perhaps act at a later phase of growth and proliferation. Alternatively, the fact that the | 

RL95-2 ceU hne is derived from a carcinoma may mean that the cell line already exhibits a ' 

maximal invasive phenotype. Consequently, it may be difficult to observe increases in the 

invasive ability of these cells after exposure to TCDD. 

Several factors should be considered in the analysis of the ability of B(a)P to 

decrease overall cellular invasiveness as determined by this assay. These factors include 

the requirement that the endometrial cells first attach to the matrigel membrane prior to 

invasion. The possibility exists that B(a)P acts at the level of cellular attachment through a 

change in the expression of ceU adhesion molecules. In this regard, we evaluated the 

ability of RL95-2 cells tireated with TCDD and B(a)P to adhere to matrigel membranes. 

The degree of attachment produced by B(a)P-treated cultures was comparable to the degree 

of overall invasion, being significantly lower than that of TCDD or control cultures. The [ 

B(a)P levels used to treat the RL95-2 cultures are on the high end of that which would ' 

present in a heavy smoker. Furthermore, only viable cells, as determined by tryphan blue 



53 

exclusion were utilized in the invasion and attachment assays, therefore the results 
observed in these experiments are most likely not the result of B(a)P cytotoxicity. 

These results are consistent with the observation that primary cells from 
endometriotic biopsies, but not normal endometrium, have been reported to exhibit the loss 
of E-cadherin expression (Gaetje et al., 1997), while a lack of p3 integrin expression was 
found to be closely correlated with a diagnosis for endometriosis (Lessey et al., 1994). In 
this regard, E-cadherin is regarded as an invasion suppressor, cell adhesion molecule. 
Secondly, the previous chapter presents evidence that B(a)P produced a state of growth 
arrest in these endometrial cell cultures under serum-free conditions, and markedly slowed 
their overall rate of prohferation in complete media. The invasion and attachment assays 
were performed after treatment of RL95-2 cultures in complete media. A reduced rate of 
proliferation produced by B(a)P could affect the ability of the cells to invade basement 
membrane if growth and proliferation are required for the elaboration of factors involved in 
the attachment/invasion process. 

Based on the simplified schematic of the process of cellular invasion (Figure 4-1), 
we next evaluated the potential of TCDD and B(a)P to alter the expression of specific 
proteases and their inhibitors which could account mechanistically for the contributions of 
these environmental agents to the etiology of endometriosis. Urokinase plasminogen 
activator (uPA) is a serine protease which catalyses the conversion of plasminogen to 
plasmin, another protease which is itself able to play a role in an array of processes such as 
tissue growth and remodeling, tumor invasion and metastasis (Mayer, 1990). The abihty 
of uPA to generate plasmin allows for the activation of members of the matrix 
metalloprotease family of enzymes capable of degrading extracellular matrix proteins like 
collagen and fibronectin (Stetler-Stevenson et al., 1993). 

Our data provide evidence that TCDD, but not B(a)P, is able to significantly 
increase the expression of steady state mRNA levels of uPA in a time- and dose-dependent 
manner. Furthermore, this induction of uPA steady state mRNA levels appeared to be the 



54 

consequence of post-transcriptional processes as shown by the ability of CHX to produce 
a superinduction of the mRNA message. In this regard, TCDD may be altering the 
expression of protein(s) which are involved in the regulation of steady state uPA mRNA 
levels. For example, the decreased expression of a protein involved in degradation of uPA 
mRNA would result in an increased half-life for the uPA message, and could account for 
the observed effect of TCDD on uPA mRNA levels. 

We next chose to evaluate whether this observable increase in mRNA was 
translated into enhanced fibrinolytic activity present in the conditioned media from 
endometrial cultures exposed to TCDD and B(a)P. Our experiments did not demonstrate 
any significant increase in the fibrinolytic activity of conditioned medium from either 
TCDD or B(a)P treated cultures. These observations led to the conclusion that if any 
differences existed, they may be present in the endometriotic tissues themselves. The fact 
that no increases in fibrinolytic activity were observed for TCDD treated cultures although 
there was a significant increase in steady state uPA mRNA expression could be the result 
of compartmentalization. Studies of cells in culture have shown that uPA can be 
compartmentalized, i.e bound to cell-surface receptors at focal contact points and remains 
active when bound to its membrane localized receptor. Hence increases in uPA activity in 
conditioned media might not be readily observed. 

These data raise a number of interesting points of discussion. First, the ability of 
TCDD to increase the steady state level of uPA mRNA has previously been demonstrated 
in human keratinocytes, which further analysis showed to be the result of post- 
transcriptional regulation via an increase in the half hfe of the uPA message (Gaido and 
Maness, 1995). Immunohistochemical analysis of the levels of PA in the endometrium of 
women with endometriosis showed a variation in the levels in normal endometrium 
throughout the menstrual cycle, whereas endometriotic tissue maintained a consistently 
high level of immunoreactive PA (Fernandez-Shaw et al., 1995). These results indicate a 
potentially more invasive nature of the endometriotic implants. 



55 

Alternatively, Malick (1982) developed the hypothesis that changes in fibrinolytic 
activity could contribute to the development of endometriosis. MaUck suggested that 
decreased peritoneal fibrinolytic activity could be responsible for the adhesions seen in the 
disease because of a decreased capacity to lyse fibrin deposits which develop secondary to 
peritoneal injury. Human endometrial cells have been shown to release two major forms 
of PA, tissue-type (tPA) and urokinase (uPA), whose expression and release are regulated 
by progesterone, estrogen and EGF (Miyauchi et al., 1995a, b). The fibrin gel 
zymography assay works equally well for the evaluation of either tPA or uPA fibrinolytic 
activity (Granelli-Pipemo and Reich, 1978). In our study, the Ij^ic activity generated by 
the conditioned medium of RL95-2 endometrial cultures is centered around a 54 kDa band 
which corresponds to the molecular weight of uPA, rather than tPA which is a 70 kDa 
protein. The fact that our data show no increase in the actual fibrinolytic activity of the 
conditioned medium is supported by other workers who have failed to find any significant 
differences in the fibrinolytic activity of peritoneal fluid from women with endometriosis 
and/or pelvic adhesive disease compared to control patients (Batzofm et al., 1985; 
Dunselman et al., 1988). 

The present study also examined the steady state level of mRNA expression of two 
members of the family of tissue inhibitors of matrix metalloproteases (TIMPs) after 
treatment of endometrial cultures with TCDD and B(a)P. TIMP-1 mRNA levels are 
increased by both TCDD and B(a)P exposure, with a greater level of induction by B(a)P 
compared to TCDD. In comparison, neither TCDD nor B(a)P significantly altered the 
levels of the two TIMP-2 mRNA transcripts. TIMPs belong to a family of proteins which 
inhibit coUagenases and gelatinases, and an imbalance between proteinases and their 
activators and inhibitors has been implicated in a number of pathological states including 
tumor invasion, fibrosis and arthritis (Stetler-Stevenson et al., 1993). Manipulation of the 
balance between MMPs and TIMPs can induce or suppress cellular invasion, as 
demonstrated by the ability of the overexpression of TIMP-2 in human melanoma A2058 



56 

cells to modulate not only proteolysis of the extracellular matrix, but also the adhesive and 
spreading properties of the cells (Ray and Stetler-Stevenson, 1995). 

The lack of change in the mRNA levels for TIMP-2 in our RL95-2 cells may not be 
at all surprising based on the recent characterization of the TIMP-2. This analysis has 
shown that TIMP-2 has several features observed in housekeeping genes, with mRNAs 
transcripts having longer half lives than that of p-actin (Hammani et al., 1996). This is in 
contrast to TIMP-1 and TIMP-3 which exhibit highly inducible levels of mRNA 
expression. The expression of TIMP-2 can be characterized as largely constitutive, in 
contrast to TIMP-1 and TIMP-3 both of which are highly inducible at the transcriptional 
level in response to phorbol esters and serum growth factors (Edwards et al., 1996). Thus 
TIMP-2 may play a major role in providing a stable basal level of inhibitory activity in 
tissues (Hammani et al., 1996). The abihty of both TCDD and B(a)P to induce TIMP-1 
mRNA levels would lend support to a reduced invasiveness for these cells, but such a 
phenomenon is observed only for B(a)P treated cultures. Cellular invasion is a multistep 
process, involving the net co-ordinated interaction of a number of genes and gene 
products. The increased TIMP-1 mRNA levels observed after TCDD and B(a)P treatment 
in this cell line may, therefore, not be readily interpretable on their own, as indicators of an 
alteration in overall cellular invasiveness. 

In summary, B(a)P, but not TCDD, was shown to inhibit the abihty of RL95-2 
endometrial cultures to attach to and traverse matrigel membranes. TCDD, but not B(a)P, 
significantly increased the steady state levels of uPA mRNA, yet neither TCDD nor B(a)P 
altered the fibrinolytic activity of the conditioned medium from treated cultures. Finally, 
both TCDD and B(a)P enhanced the level of expression of TIMP-1 mRNA, but had no 
effect on TIMP-2 expression. Further work needs to be carried out in order to better 
characterize the overall effect of these environmental agents on cellular invasion and tissue 
remodeling factors and so elucidate their full potential role in uterine pathologies. 



57 



Plasmin 



Plasminogen 




BASEMENT MEMBRANE 



ENDOMETRIAL CET J, 



Figure 4-1. Illustration outlining the potential mechanism of invasion and 
implantation of endometrial cells. 



(A) 



58 



, ^^---^hiibi^t- 



<«■*'_ ..f. 



(B) 










> * . f 









H«rtliili«4lltu»,._^ 



Figure 4-2. Diagram of a modified Boyden Chamber apparatus. 
(A) Cross-sectional view and (B) View fron above. 



59 



150 -, 



2 

u 

On 

u 

O 

o 



100- 



El3 Control 



■ BaP (10 |iM) 
□ TCDD (10 nM) 



1 




Figure 4-3. Quantitation of the effect of 10 nM TCDD and 10 i^M B(a)P 
pre-treatment on the ability of RL95-2 cells to invade Matrigel membranes. 
Approximately 2 x lO'* cells were aliquoted into the bottom wells of the 
apparatus and allowed to attach to a 8 micron Matrigel-coated membrane for 90 
min after inversion. Upper wells were loaded with complete media and cells 
allowed to migrate for 36 hr. Cells which migrated through the membrane 
were stained with Leukostat and counted under a light microscope. Data are 
expressed as the mean ± SEM of the number of migrating cells from three 
separate experiments. * P < 0.001 as compared to 0.1% DMSO controls. 



60 



(A). 



Control 



B(a)P 

(10 |iM) 



TCDD 
(10 nM) 



% # 



'4^ 



(B). 



2 

u 

p— N 

o 

• 

o 



500 -, 



400- 



300- 



200- 



100- 




T 



\ \ \ \\\ \ -H •■ 

jf y y r jf jf jT 
\ \ \ \1a. \ \ 

y j^ y j^ jT y y 
\ -^ \ \ \ \ \ 

y y y y y y y 
\ \ \ '^ \ \ \. 
y y y y y y y 

\ \ \ \ \ -^ 
y y y y y y y 

\ \ \ \ \ \ 
y y y y y y y 

\ "^ \ \ \ \ 
y y y y y y y 

\ \ \ \ ••. \ 
y y y y y y y 

\ \ \ \ \ \ 
y y y y y y y 

\ \ \ \ \ \ 
y y y y y y y 

\ \ \ \ \ \ 
y y y y y y y 

\ \ \ \ \ \ ■ 
y y y y y y y 

\ \ \ \ \ \ • 
y y y y y y y 

'■. \ \ \ \ \ ■ 
y y y y y y y 

\ \ \ \ \ \ • 
y y y y y y ji- 

\ s \ \ \ \ ' 
y y y y y y y 

\ \ \ \ X \ ' 
y y y y y y y 

\ \ \ \ \ \ ■ 
y y y y y y y 

\ \ \ \ \ \ • 
y y y y y y y 
. \ \ \ \ \ \ • 
y y y y y y y 

\ \ \ \ \ \ • 
y y y y y y y 

\ \ \ \ ■-. \ ■ 
y y y y y y y 

\ \. \ \ \ \ • 
y y y y y y y 

\ \ \ \ \ \ • 



H Control 
■ 10 |iM BaP 
S 10 nM TCDD 



Figure 4-4. Quantitation of the effect of 10 nM TCDD and 10 jiM B(a)P 
pre-treatment on the ability of RL95-2 cells to attach to Matrigel membranes. 
Approximately 5x10^ cells were aliquoted into the bottom wells of the 
apparatus and allowed to attach to a 8 micron Matrigel-coated membrane for 2 
hr after inversion. Cells which attached to the membrane were stained with 
Leukostat and counted under a light microscope. (A) Scanned image of 
treated RL95-2 cell attached to matrigel membranes. (B) Data expressed as 
the mean ± SEM of the number of attached cells from two separate 
experiments. * P < 0.001 as compared to 0.1% DMSO controls. 



61 



(A) 



6h 12h 24h 48h 



TCDD (10 nM) - + - + - + - + 



P-Actin 




(B) 



B(a)P (10 |iM) 
uPA 



1 10 



• igl^ Jlll~ 



p-Actin 






Figure 4-5. Northern blot analysis of uPA mRNA. Cells were exposed to TCDD or 
B(a)P and total RNA isolated, denatured, blotted and hybridized with 32P-labeled 
cDNA probes as described in Materials and Methods. (A) Representative blot of the 
time-dependence of the induction of uPA with 10 nM TCDD treatment. (B) Blot 
of the effect of B(a)P exposure on uPA mRNA levels in RL95-2 cells for 48 hr. 



62 



(A). 



48h 

TCDD(nM) 0.1 1 10 
uPA 

P-ACTIN 




(B). 



1.5 



> 

'■a 

^ 2 

^ s 

:« o 

w o 

J3 ^ 



O 



1- 



0.5 



0- 



-©- 



TCDD (10 nM) 
TCDD (1 nM) 
TCDD (0.1 nM) 



> 








I 
10 



20 



30 



40 



SO 



TIME (h) 



Figure 4-6. Dose-dependent effect of TCDD on the expression of uPA 
mRNA in RL95-2 cells. (A) Northern blot analysis of total RNA after a 48 
hr exposure to varying concentrations of TCDD. Analysis performed as 
described in Materials and Methods. (B) Graphical representation of the 
dose-dependent effect of TCDD on uPA mRNA levels. Data represent the 
mean scan intensity ± SEM of at least three experiments. * P < 0.05. 



63 



(A) 



PLASMINOGEN 



uPA (IN GEL LANES) 



FIBRINOGEN 




FIBRIN 



FIBRIN DEGRADATION 
PRODUCTS (Lysis) 



(B) 




Figure 4-7. Evaluation of the fibrinolytic activity of the conditioned media from 
B(a)P and TCDD treated RL95-2 cultores. (A) An outline of the methodology 
mvolved m the fibrmolytic analytical technique. (B) Representative photograph 
of the lytic bands produced by the plasminogen activity of the conditioned media 
from the treated cultures. 



64 



(A). 



# # # 






C ^ *■- <^v 



TIMPl 



P-ACTIN jff} 




(B). 





500- 






400- 




$3 






cs 






^i^ 


300- 




• l-N 






tive 
tens 


200- 




a ffl 






^-N h^ 






CD 






Pi5 


100- 





Control 
m TCDD (10 nM) 

a B(a)P (1 ^iM) 
13 B(a)P (10 ^iM) 




Figure 4-8. Northern analysis of TIMP-1 mRNA. RL95-2 cultures were exposed to 
TCDD, B(a)P or 0.1% DMSO for 48 hr after which total RNA was isolated, 
denatured, blotted and hybridized to 32P-labeled cDNA probes as described under 
Materials and Methods. (A) Representative Northern blot probed with 32p-TIMP-l. 
(B) Graphical representation of the scan intensity from treated cultures. Bars represent 
the mean ± SEM of at least three experiments. * P < 0.05 



(A) 



65 



TCDD (10 nM) 
TIMP2 

TIMP2 




\'' * -^ ^"'"^ ^^^'• 



P-ACTIN 



UK ^0 




"Sat 



B.. 



(B) 



12h 



24h 



48h 



B(a)P (10 M,M) ^. 
TIMP2 




TIMP2 



P-ACTIN 



Figure 4-9. Northern analysis of TIMP-2 mRNA. RL92-2 cultures were exposed 
to (A) 10 nM TCDD, (B) 10 ^M B(a)P, or 0. 1% DMSO for 48 hr and total RNA 
isolated, denatured, blotted and hybridized to 32p-iabeled cDNA probes as 
described under Materials and Methods. (A) and (B) are representative Northern 
blot probed with 32P-TIMP-2 generated from cultures treated as indicated Blots 
show the presence of both the 1.2 and 3.5 kb mRNA transcripts for TIMP-2. 



CHAPTER 5 
EFFECTS OF TCDD ON IL-lp AND TNFa IN A HUMAN ENDOMETRIAL CELL 

LE^ 



Introduction 

Normal endometrium undergoes predictable biochemical and histological changes in 
response to hormones throughout the menstrual cycle. Cytokines including IL-1 and 
TNFa are produced by cell populations within the uterine environment and may participate 
in growth and differentiation of the endometrium (Frank et al., 1995; Laird et al., 1996; 
Roby and Hunt, 1994; Simon et al., 1993). IL-1 and TNFa are among the pleiotropic 
growth factors which may act as local mediators of cellular communication in the uterine 
cavity (Hunt et al., 1992), with the IL-1 system having recently been demonstrated to play 
a role in the process of embryonic implantation (Simon et al., 1994). Dysregulation of the 
expression of these cytokines could potentially result in pathological disorders of the 
endometrium and uterine cavity and musculatare. 

The Sampson hypothesis postulates that endometriosis is a consequence of the 
implantation and growth of desquamated endometrial cells and fragments at extrauterine 
sites, yet this model does not take into account the prevalence of retrograde menstruation 
within the female population (Halme et al., 1984). An altemative theory is based on 
evidence that alterations in the normal immune system regulation may facilitate the 
implantation of endometrial fragments, thereby contributing to growth, and disease 
progression (Dmowski et al., 1994; Gleicher and Pratt, 1993; Rier et al., 1995). Evidence 
in support of this immunologic hypothesis comes from data showing evidence of elevated 
levels of inflammatory ceU products such as IL-1, IL-5, IL-6, IL-8, IL-10, TNF-a and 
prostaglandins in peritoneal fluid as well as macrophage and macrophage-conditioned 

66 



67 

media from endometriosis patients (Keenan et al., 1995; Koyama et al., 1993; Mori et al., 
1991; Rana et al., 1996; Taketani et al., 1992). In this regard, danazol, a mainstay of the 
medical management of endometriosis, is able to suppress the production of IL-1 and TNF 
by human monocytes (Mori et al., 1990). Further evidence for a role for immunological 
factors in the maintenance of endometriotic implants is based on the ability of macrophages 
to secrete cytokines capable of influencing endometrial growth, as well as the observation 
that peritoneal fluid from women with endometriosis increases endometrial cell proliferation 
(Ramey and Archer, 1993). Alterations in cell-mediated and humoral immunity have also 
been noted in the disease (Dmowski et al., 1994; Gleicher, 1994). 

TCDD is a well characterized immunotoxicant (Holsapple et al., 1996; Kerkvliet, 
1995; Masten and Shiverick, 1995). TCDD has previously been shown to induce the 
expression of both IL-lp and TNFa in vivo (Fan et al., 1997) and in vitro in human 
keratinocytes (Sutter et al., 1991) and in MCF-7 breast cancer cells (Vogel and Abel, 
1995). The ability of these compounds to alter cytokine expression could facilitate a role in 
the pathogenesis of uterine disease. The objective of this study was to evaluate the effects 
of TCDD on the expression of IL-1 (3 and TNFa in a human endometrial cell culture model. 
The study examined the dose and time dependence of TCDD exposure with changes in 
cytokine mRNA levels. 

Results 

Effects of TCDD on IL-lg and TNF-a mRNA levels in RL95-2 Cells 

TCDD exposure significantly increased the steady state level of mRNA for the IL- 
1(3 transcript relative to controls as evaluated by Northern blot analysis (Figure 5-1). Data 
show that 10 nM TCDD exposure of RL95-2 cultures resulted in a time-dependent 
induction of the expression of IL-1 (3 mRNA which is observed as early as 6 hr after 
treatment. Maximal induction of about 5-fold occurred by 36 hr after TCDD treatment. 



68 

with levels remaining elevated above controls for the duration of the 48 hr assay period 
(Figure 5-2). Not only was the induction of IL-lp mRNA time-dependent, but it was also 
observed to be dose-dependent (Figure 5-lB). A representative Northern blot performed 
after a 24 hr treatment with concentrations of TCDD from 0.1-10 nM TCDD indicates that 
IL-lp mRNA is induced by TCDD exposures as low as 0. 1 nM and is maximal by 10 nM 
TCDD. In comparison, RL95-2 exposure to TCDD led to a significant (p < 0.05), time- 
dependent induction of TNF-a mRNA steady state levels by 36 hr (Figure 5-3). Levels 
plateaued at this 36 hr time point and remained elevated relative to controls for the 
remainder of the 48 hr assay period (Figure 5-3B). Hence TCDD treatment resulted in 
significantly increased levels of mRNA for both IL-ip and TNF-a. However, the time 
course and relative levels of induction varied between both messages. IL-ip mRNA was 
significantly increased at an earlier time point as well as showing higher levels of induction 
at maximal levels relative to TNF-a (Figure 5-2 and 5-3). It warrants note that the late 
induction of TNF-a showed a time-dependency similar to that exhibited for uPA induction 

(Figure 4-5 and 5-3). | 

I 

'I 

1; 

Effect of TCDD on the Rate of Transcription of CYP1 AL CYP1B1. uPA. and n.-1J ^ 
mRNA in RL95-2 Cells 

Nuclear runoff analyses were performed in order to determine whether the observed 
changes in uPA and IL-lp mRNA levels were the result of increased transcriptional 
activity. Nuclei were isolated from RL95-2 cells treated with 0.1% DMSO and 10 nM 
TCDD and analyzed for the levels of nascent mRNA transcripts. Data in Figure 5-4 
demonstrate that CYPlAl transcription is significantly increased 5-fold by TCDD, whereas 
no effect was observed on CYPIB 1, uPA or IL-lp transcripts. 

Furthermore, we also utilized the protein synthesis inhibitor cycloheximide (CHX) 
to investigate whether the induction of uPA, TNF-a and IL-ip mRNA was dependent upon 



69 

the synthesis of other proteins. As shown in Figure 5-5, the exposure of cultures of RL95- 
2 cells to CHX in the presence or absence of TCDD led to the superinduction of uPA, 
TNF-a and IL-lp mRNA levels, implying that the levels of these mRNA are tightly 
regulated and dependent upon de novo protein synthesis. Gaido et al (1995) has recentiy 
shown that TCDD does in fact regulate the level of uPA mRNA in a human keratinocyte 
cell line via a post transcriptional mechanism. Thus evidence indicates that TCDD is not 
acting to increase the steady state levels of mRNAs for IL-lp, TNF-a and uPA by directly 
altering the rate of mRNA transcription. 

Discussion 

The present study investigated the potential of TCDD to alter the expression of 
cytokine/growth factor genes which may play a role of the etiology of endometriosis. Our 
data indicate that TCDD is able to induce the expression of mRNA for both IL-lp and 
TNF-a in the RL95-2 cell line. In the case of IL-ip, this induction is both time- and dose- 
dependent. For TNF-a, a time-dependent increase in steady state levels of mRNA was 
observed with TCDD exposure. This induction of IL-lp and TNF-a by TCDD has been 
demonstrated previously in human keratinocytes (Sutter et al., 1991), a breast cancer cell 
line (Vogel and Abdel, 1995) and, more recently, in rat hepatic tissue after in vivo exposure 
(Fang et al., 1997). Significantly, this is the first observation of these responses in uterine 
cells and may lead to a better understanding of the potential actions of TCDD and related 
compounds in uterine disease etiology. 

IL-ip and TNF-a are pleiotropic cytokines secreted by a variety of cell types. Their 
role in mediating responses in the female reproductive system and during the course of 
gestation has recently come under greater scrutiny (Simon et al., 1994; Tabibzadeh, 1991). 
Many of the processes occurring during menstruation are reminiscent of the inflammatory 
response in terms of cellular proliferation, ischemic necrosis, stromal granulocyte 
infiltration and angiogenesis (Tabibzadeh, 1991). Both cytokines have been detected in 



70 

human and rodent uterine tissue, with their expression apparently being cyclically regulated 
by estrogen and progesterone levels (Frank et al., 1995; Hunt et al., 1992; Laird et al., 
1996; Roby and Hunt, 1994). The pleiotropic nature of these polypeptides, has generated 
difficulty in defining their precise roles in the uterine environment. 

The immunological hypothesis of endometrial promotion, has resulted in study of 
IL-ip and TNF for their potential contribution to the pathology of the disease. Increased 
numbers of activated macrophages have been detected in the peritoneal and mbal fluids of 
infertile women with endometriosis (Haney et al., 1983). Activated macrophages secrete 
IL-lp, and increased levels of this cytokine have been found in the peritoneal fluid of 
women with endometriosis above that from healthy women or those treated for the disease 
(Keenan et al., 1995; Mori et al., 1991; Rana et al., 1996). Danazol, one of the mainstays 
of the medical management of endometriosis, results in decreased levels of these cytokines 
in the peritoneal fluid of treated subjects (Mori et al., 1990; Taketani et al., 1992). 

The outcome of an inflammatory cascade could lead to the synthesis of new 
connective tissue and adhesion formation as weU as increased vascularity as the result of 
angiogenesis, processes which could be mediated by activated macrophages through their 
elaboration of pro-inflammatory cytokines. Induction of angiogenesis, procoagulant 
activity and mitogenic action on fibroblasts are among the actions of IL-lp and TNF 
(Tabibzadeh, 1991). IL-1 has been shown to stimulate collagen deposition and fibrinogen 
formation (Posthlewaite et al., 1984), which might account for the incidence of fibrosis and 
adhesions observed in advanced stages of endometriosis. Peritoneal fiuid from 
endometriotic women has been shown to have a toxic effect on mouse embryo development 
(Taketani et al., 1992), an observation which has been reproduced with IL-ip (Fakih et al., 
1987), although some controversy is involved in the reproducibility of this data (Schneider 
et al., 1989). 

The data we have presented demonstrates a time-dependence of the induction of 
messages for both IL-1 [3 and TNF, with IL-ip induction being observed by 6 hr and TNF- 



71 



a being apparent by 36 hr. This observation is interesting in light of the fact that IL-lp has 
been shown to stimulate the production of TNF-a in endometrial cells and cytotrophoblasts 
in culture (Knofler et al., 1997; Laird et al., 1996). It has been suggested that cytokines 
are integrated into an intricate network that operates by coordinated regulation of their 
expression. Our observation of the time-dependent induction of mRNA levels for IL-1(3 
and TNF may be a manifestation of this phenomenon. 

The fact that IL-lp and TNF are characterized primarily as immune system 
modulators, supports their potential role in the pathogenesis of uterine disorders like 
endometriosis and may relate to effects on cellular attachment and invasion processes. IL- 
lp has recently been shown to be involved in the ability of murine embryos to be 
successfully implanted (Simon et al., 1994), while the adherence of endometrial stromal 
cells to mesothelial cells was significantly increased by pretreatment of mesotheUal cells 
with TNF-a (Zhang et al., 1993b). The latter observation may be associated with the 
abihty of TNF to alter the expression of molecules involved in adhesion interactions such 
as cadherin and p-catenin (Tabibzadeh et al, 1995). The role of these pleiotropic cytokines 
in the process of implantation is further supported by their ability to alter the expression of 
matrix metalloproteases (MMPs), enzymes involved in the degradation of basement 
membrane molecules to allow for tissue remodeling (Librach et al., 1994; Sato et al., 
1996). 

The present study also examined the possibility that the induction of mRNA for 
these cytokines could potentially be regulated at the transcriptional level. To this end, we 
utilized the method of nuclear runoff analysis to examine the translation of nascent 
transcripts from TCDD-treated cultures of RL95-2 cells. Our data indicate that while 
CYPlAl steady state mRNA levels were indeed increased as a result of increased 
transcription rates, IL-lp and uPA mRNA levels were not (Figure 5-4). Both IL-1 and 
TNF have been reported to be regulated at both the transcriptional and post-transcriptional 
level (Fenton, 1992; Tabibzadeh, 1991). Data in Figure 5-5 which show the 



72 

superinduction of IL-1[3 mRNA levels after exposure of RL95-2 cultures to CHX, a protein 
synthesis inhibitor, lends support to a post-transcriptional mechanism of regulation. TNF- 
a, for example, is thought to be primarily controlled at the post-transcriptional level, and 
cells expressing the TNF-a message may not necessarily translate it into product (Sariban 
et al., 1988). In fact, TNF-a release from cytotrophoblast cultures was demonstrated to be 
independent of the induction of its mRNA levels (Knofler et al., 1997). 

In summary, TCDD induced the expression of the steady state levels of mRNA for 
IL-lp and TNF-a in a time-dependent manner. Analysis of the transcription of IL-1|3 
demonstrated that the induction was not a result of increased transcription rates, but likely 
the consequence of post-transcriptional phenomena. Thus, chronic inflammatory changes 
in endometriosis may be mediated at least in part by IL-lp and TNF-a. Our data showing 
increased expression of IL-ip and TNF-a mRNA after TCDD exposure opens the 
possibility TCDD may also play a role not only in the invasive potential of desquamated 
endometrium, but also in the infertility associated with the disease. 



(A) 



73 



6h 12h 24h 36h 48h 



TCDD (10 nM) 

IL-lp 

p-Actin 



+ - + -+- + - + 



(B) 



3(ih 



TCDD (nM) 





0.1 1 10 


IL-lp 
P-ACIIN 


i 


1,:-*** 



Figure 5- 1 . Northern blot analysis of IL- 1 p showing the dose- and time-dependence 
of its induction with TCDD. (A) Representative Northern blot showing the time- 
dependence of the induction of IL-lp mRNA with 10 nM TCDD exposure. (B) A 
representative Northern blot showing the dose-dependence of IL-lp mRNA induction 
after a 36 hr exposure to varying concentrations of TCDD. Forty ng of total RNA 
was denatured, blotted and hybridized with 32P-Iabeled cDNA as described under 
Materials and Methods. 



74 



fi 




C3 




O 


>■ 


^ 


-*-» 




• pH 


> 

tm4 




■^^ 


■4-* 


a 


fl 




hH 


a> 




P^ 









/ 
/ 
/ 
/ 


: 1 — >• — 1 


150- 

100- 

50- 


^ Control 

--^-- IL-ip 

/ 

/ 

/ 

/ 

*A" -L 






— A 

1 


0- 


1 -1 



10 20 30 40 

TIME (h) 



50 



Figure 5-2. Graphical analysis of the induction of IL-lp mRNA in 
RL95-2 cultures treated with 10 nM TCDD (A), relative to 0.1% 
(v/v) DMSO (x) controls. Data represent the mean ± SEM from five 
experiments. * P< 0.001 compared to controls. 



75 



(A) 



36h 



48h 



TCDD (lOnM) 

TNF-a 

J3-ACTIN 




(B) 



as 

U 

2 



< 

E 

tin 

Z 



1.5- 




0.5- 



30 40 50 



TIME (h) 



Figure 5-3. The effect of TCDD exposure on TNF-a mRNA expression in RL95-7 
£5''^-J^^ Northern analysis of TNF-a showing a time-dependent induction with " 
TCDD exposure. Total RNA. 40 ^ig, was denatured, blotted and hybridized with '^P 
labeled cDNA for TNF-a as described in Materials and Methods. (B) Graphical 
analysis of the induction of TNF-a mRNA with time of exposure to TCDD Values 
represent the mean ± SEM of three experiments. * P < 05 



76 



(A) 



T 



uPA 






IL-lp 

CYPlAl 
CYPIBI 



p-ACTIN 



"Sff 



(B) 






Pi 

o 



H 




H uPA 

g IL-ip 

CYPlAl 

[□ CYPIBI 



Figure 5-4. Effect of TCDD on the rate of IL-lp and uPA transcription. Actively 
proliferating RL95-2 cultures were treated with either 10 nM TCDD (T) or 0.1% 
(v/v) DMSO (C). Nuclei were isolated after 44 hr and nuclear runoffs performed 
as described under methods, p-actin was used as a loading control and CYPlAl 
as a positive control for transcriptional induction by TCDD. (A) Representative 
autoradiogram of a runoff analysis for transcriptional induction. (B) Results of 
the desitometric analysis of the band intensities after normalization to P-actin 
indicating relative changes in rates of transcription. Each bar represents the 
mean ± SEM of at least three experiments for 10 nM TCDD treatment. 
* P< 0.0] compared to p-actin. 



77 



CHX (5 |ig/ml) 
TCDD (10 nM) 

uPA 



TNF-a 




IL-lp 



"wy 



P-Actin 




Figure 5-5. Northern analysis showing superinduction of mRNA for uPA, 
TNF-a and IL-lp by cycloheximide in RL95-2 cells. Fifty |ig of total RNA 
from RL95-2 cultures was isolated after 12 hr treatment with the protein 
synthesis inhibitor cycloheximide (CHX), in the presence or absence of 10 
nM TCDD. Bands were visualized by autoradiography after membranes were 
probed with "P-labelled cDNA probes. 



CHAPTER 6 

EXPRESSION AND PURIFICATION OF RECOMBINANT AND NATIVE 

PROLACTIN-LIKE PROTEE>^S B AND C 



Introduction 

The rodent placenta is a rich source of proteins which are structurally related to 
pituitary PRL. To date, fifteen of these PRL-like proteins have been identified and shown 
to exhibit a temporal and cell-specific pattern of expression. It has been suggested that 
these proteins are members of a superfamily of hormones and cytokines, the helix-bundle- 
peptide (HBP) hormones, with shared structaral features and mechanisms of action. The 
HBP structure consists of four long a-hehces arranged in antiparallel fashion (Horseman 
and Yi-Lee 1994) with family members including GH, PRL, erythropoietin and the 
interleukins. Based on their homology to PRL, some of these placental proteins have been 
characterized and shown to possess PRL-like bioactivity (Cohick et al., 1995; Colosi et al., 
1987a, b; Deb et al., 1991a; Robertson et al, 1982, 1994). The biological activity of a 
number of these proteins, however, remains as yet undetermined. 

Our laboratory had previously identified four major rat placental proteins secreted 
into conditioned media from placental explant cultures. These proteins were identified by 
N-terminal sequence analysis to be related to the PRL/GH family (Ogilvie et al., 1990a). A 
cDNA coding for a protein virtually identical to that of Proteins 2 and 4 was later isolated 
by Deb et al (1991b) and designated PLP-C based on an akeady established nomenclature 
(Duckworth et al., 1986b, 1988). Our laboratory has studied several models of low birth 
weight in pregnant rats and found that maternal xenobiotic exposure and protein 
malnutrition, respectively, are associated with decreased placental growth, vascular 
development and intrauterine growth retardation (lUGR). Moreover, growth-retarded 



78 



79 

placentas showed decreased expression of protein and mRNA for PLP-B (ConliflFe et al., 
1995; Shiverick et al., 1991). Thus, previous evidence supports an association between 
impaired placental growth and decreased expression of PLP-B and PLP-C proteins. 

In an effort to identify the biological role of PLP-B and PLP-C in placental growth, 
our laboratoiy initiated efforts to purify native forms of the placental protein, as well as 
attempt recombinant expression of PLP-B and PLP-C proteins in both bacterial and 
mammalian systems. This section of the study details the expression and purification of 
recombinant PLP-C using two different bacterial constructs, as well as the mammalian 
expression and purification of PLP-B. Recombinant PLP-C was further used to generate a 
polyclonal antisera which was essential for immunoaffmity purification of the native protein 
from conditioned media ofbasal zone explant cultures. Finally, this section also details the 
use of an antipeptide antisera previously generated against the C-terminus of PLP-B 
(Ogilvie et al., 1990b) for purification of native protein. 

Results 
Purification of Recombinant hCAII-PLP-C 

In the hCA-n fusion vector p0304 (Van Heeke et al., 1993), the DNA sequence 
encoding the recombinant protein is linked to hCA-II through a cleavage recognition 
sequence for enterokinase. In order to achieve the expression and subsequent release of 
PLP-C from the hCAII fusion protein, it was necessary to subclone the DNA fragment 
encoding PLP-C into an EcoRV site located adjacent to the enterokinase cleavage site such 
that a DmI site would be present immediately adjacent to the codon specifying the first 
amino acid in the mature form of PLP-C (Conliffe et al., 1994). Aliquots from different 
stages in the purification of recombinant PLP-C were separated on 10% SDS-PA gels 
under reducing conditions and proteins were visualized by Coomassie staining (Figure 6- 
1). The predicted molecular weight of the hCAII-PLP-C fusion protein is 53,620 (hCAII, 



80 



MW 31,234; PLP-C, MW 22,386). As shown in Figure 6-1, lanes A and D, a band 
corresponding to a protein of 53 kDa was purified from the bacterial extract. The linkage 
of PLP-C to hCAQ allowed for rapid purification of the fusion construct utilizing a pAMBS 
resin with high affinity for hCAII. The 53 kDa band comprised approximately 15% of the 
Coomassie blue-stained affmity-purified protein (Figure 6-1, lane D). 

Western immunoblot analysis using a PLP-C antipeptide antisera revealed a broad 
band of 31-36 kDa and another band of 53 kDa in the cell lysate and pAMBS-purified 
fusion protein preparations (Figure 6-2, left panel, lanes A and D.). The immunoreactive 
protein of 53 kDa corresponded to the calculated molecular weight of the fusion construct 
of hCAII-PLP-C and also cross reacted with hCAII antisera (Figure 6-2, right panel, lane 
D). The broad 31-36 kDa immunoreactive band comprised about 75% of the purified 
fusion protein and the molecular weight was confirmed by Mass Spectrometry to be 31-36 
kDa. These lower molecular weight bands were initially thought to be hCAII-PLP-C 
proteolytic products resulting from the action of bacterial proteases released during ceU 
lysis. The addition of the protease inhibitors, phenylmetiiylsulfonyl fluoride, leupeptin, 
aprotinin and EDTA during cell lysis, however, did not reduce the staining intensity of 
these bands (data not shown). These bands also stained positively with a hCAII antisera 
(Figure 6-2, right panel, lanes A and D) and had an N-terminal sequence, His-His-Trp- 
Gly-Tyr-Gly-Lys-His-Asn-Gly, which was identical to that of hCAII. Thus, data indicate 
that the immunoreactive bands of 53 kDa, as well as the 31-36 kDa were hCAII fusion 
proteins. 

Enterokinase cleavage of the affinity-purified fusion protein produced an 
immunoreactive protein band of approximately 25 kDa (Figure 6-3, lanes A and B), 
corresponding to the calculated molecular weight of PLP-C. As judged by Coomassie blue 
staining, the 25 kDa protein was highly enriched by affinity purification (Fig. 6-3, lane B). 
Recombinant PLP-C yield varied with different preparations. Enterokinase cleavage of 
PLP-C from its fusion partner produced approximately 15% recombinant PLP-C. Efforts 



81 

were made to maximize cleavage conditions, by increasing enzyme concentration and 
incubation times, as well as by changing Ca2+ concentration, but there was not a significant 
increase in yield of PLP-C protein. 

Recombinant Expression and Purification of pET22b('+)His-PLP-C 

Because of the poor yield of the original hCAII-PLP-C construct in generating 
recombinant PLP-C, we undertook the construction of an alternative bacterial expression 
system (Figure 6-4). Expression of recombinant PLP-C with a polyhistidine tag at its N- 
terminus was chosen because this system allowed for purification using the ability of poly- 
histidine to bind divalent metal ions even under denaturing conditions (Hochuli et al., 
1987). To achieve the expression and subsequent release of PLP-C protein from the 
pET22b(+) expression system, it was necessary to anneal a polyhistidine primer to the 5' 
end of the PLP-C cDNA from the original hCAII-PLP-C vector which included the intact 
enterokinase linker region. The resulting construct was then hgated into the NdellNotl 
sites of the pET22b(+) expression vector as described under Materials and Methods and as 
outlined in Figure 6-4. The presence and orientation of the desired insert was determined 
by restriction enzyme digestion with Ndel which would result in the release of an 
approximate 700 bp fragment of the PLP-C cDNA as shown in Figure 6-5. 

Aliquots from diiferent steps in the purification of recombinant PLP-C from the 
pET22b(+)His-PLP-C system were separated by 10% SDS-PAGE under reducing 
conditions and proteins visualized by Coomassie staining (Figure 6-6). The recombinant 
protein of approximately 27 kDa is seen in lanes A , D, E, G and H of Figure 6-6A. The 
figure shows steps from two different purification schemes. Lanes A-D are from the 
soluble fraction of the bacterial pellet. The recovery of mostly insoluble protein (lanes E- 
H) required detergent solubilization of the pellet in N-laurylsarcosine (detergent), followed 
by refolding under alkaline conditions at room temperature overnight (Luck et al., 1991, 



82 

1992). The His-PLP-C protein was then isolated from the supernatant by affinity 
purification on immobilized nickel cation columns. Figure 6-6B is a Coomassie stained gel 
which illustrates the results of the enterokinase cleavage of the His-PLP-C protein to 
generate the full length recombinant protein. Lane A represents the full length protein after 
enterokinase cleavage of the His-PLP-C product (Lane B). The 25 kDa protein was 
electroblotted onto PVDF membrane and amino terminal sequence analysis revealed a major 
sequence of Ile-Pro-Ala-Cys-Met-Val-Glu. This sequence was identical to that of amino 
acids 1-7 of PLP-C (Deb et al., 1991b, c; Ogilvie et al., 1990b). A minor species, less 
than 25%, was also detected, with N-terminal sequence corresponding to residues 6-12 of 
PLP-C. 

Expression of Recombinant PLP-B 

The expression of reconbinant PLP-B in a mammalian system was chosen because 
of the fact that the native protein exists only as a glycosylated species (Duckworth et al., 
1986b). The complete cDNA coding sequence for PLP-B was blunt-end Ugated into the 
pMXSND expression vector as outlined in Figure 6-7. This vector has previously proven 
useful in the generation of recombinant proteins for other members of the PRL/GH family 
(Cohick et al., 1997; Deb et al., 1993; Lee and Nathans, 1988). Blunt-ended ligation of 
the PLP-B cDNA was necessary because there was only a single Xhol cloning site in the 
pMXSND vector with no other compatible site for 'sticky ended' ligation with the PLP-B 
cDNA. 

BamHl diagnostic restriction endonuclease digests were performed to determine the 
presence of the PLP-B insert in the correct orientation. Figure 6-8 illustrates the results of 
digests performed upon putative plasmids containing the PLP-B insert. Lanes 8 and 10 
show results of the diagnostic digests of plasmids incorporating the pMXSND-PLP-B 
insert in the incorrect and correct orientation, respectively. CHOKl cells were transfected 
with the pMXSND-PLP-B plasmid and stable transfectants selected by culture in the 



83 

presence of G418. Stable clones were selected after expansion by probing for the presence 
of mRNA species which hybridized to ^^P-PLP-B cDNA on Northern blot analysis (Figure 
6-9). Clones 4, 5 and 7 were observed to give positive hybridization signals and were 
consequently expanded by growth in large cultures with G418 and MTX selection. PLP-B 
secretion was induced by the culture of stable transfectants in the presence of CdClj which 
makes use of the metallothionein promoter in the pMXSND vector. Western immunoblot 
analysis of concentrated conditioned media from these stably transfected cultures was 
performed using an antipeptide antisera against the C-terminus of PLP-B (Ogilvie et al., 
1990b) and demonstrated the presence of immunoreactive protein of approximately 30 kDa 
(Figure 6-10) which corresponded in size to native PLP-B protein (Ogilvie et al., 1990a). 

Purification and Western Immunoblot Analysis of Native PLP-B and PLP-C 

Native PLP-B and PLP-C were purified from the conditioned media of gestation 
day 18 rat placental (basal zone) explant cultures. The utility of antisera generated against 
these proteins (Ogilvie et al., 1900b; Conliffe et al., 1994) in immunoafFinity 
chromatography was demonstrated. The purified native preparations of PLP-B and PLP-C 
were characterized by two dimensional SDS-PAGE. As seen in Figure 6-11, PLP-B is 
present as a series of 30 kDa subforms with pi ranging between 6.1-6.6, as observed by 
silver staining (Figure 6- 11 A) and Western immunoblot analysis (Figure 6-llB). The anti- 
PLP-C column retained species of 25 kDa and 29 kDa with pi ranging from 5.8-6.2, as 
observed on silver stained two dimensional SDS-PAGE (Figure 6-12A) and confirmed by 
Western immunoblot analysis (Figure 6-12B). These data are in agreement with the 
previously reported size and pi characteristics of secreted PLP-B (Ogilvie et al., 1990a) and 
PLP-C (Conliffe et al., 1994). 



84 
Discussion 

The present study describes the recombinant expression and purification of PLP-B 
and PLP-C, two members of the PRL-GH family produced by the rat placenta. 
Furthermore, the study also describes the purification of these native proteins from 
placental explant cultures by immunoaffinity chromatography with the use of antibodies 
generated against PLP-B and PLP-C. Recombinant PLP-C was initially expressed as a 
fusion partner with human carbonic anhydrase (hCAII) in JM109(DE3) E. coli bacteria. 
The usefulness of hCAII has been previously demonstrated as a partner in fusion protein 
expression constructs with E.coli Fl-ATPase e subunit and asparagine synthethase A, 
respectively, (Hinchman and Schuster, 1992; \kn Heeke et al., 1993). In the present 
study, linkage of PLP-C to hCAII allowed the fusion protein to be purified to homogeneity 
in a single step on a sulfonamide affinity resin. The desired 53.4 kDa fusion protein was 
produced in our expression system in addition to several other hCAII fusion proteins of 31- 
36 kDa. These lower molecular weight proteins may represent premature translation 
termination products of PLP-C. In this regard, it is possible that chain termination might 
be due to differences in codon usage between mammalian and bacterial systems. 

In the expression system for the hCAII fusion protein, the recombinant PLP-C 
protein is released from its fusion partner by enterokinase cleavage. Although enterokinase 
digestion of hCAII-PLP-C released recombinant PLP-C, the yield was found to be highly 
variable. Because the enterokinase products of the 31-36 kDa bands would not exceed 6 
kDa, it is expected that they are excluded from the preparation during the centriprep 
concentration step which has a 10 kDa cutoff membrane. In this regard, Hinchman and 
Schuster (1992) were unable to obtain any cleaved product in the expression of asparagine 
synthethase A. In contrast, release of PLP-C from the His-PLP-C construct was achieved 
with a high level of consistency. This disparity in release of the recombinant protein may 
be attributed to differences in enterokinase preparations or, alternatively, to folding of the 
recombinant protein which might interfere with cleavage. 



85 

The difficulties encountered in the use of the hCAII fusion system for the 
expression of recombinant PLP-C necessitated the development of an alternative expression 
construct. The enterokinase PLP-C sequence was excised from the original hCAII-PLP-C 
construct and ligated to a polyhistidine tract at the N-terminal region of PLP-C upstream of 
the enterokinase region. The presence of the polyhistidine region allowed for purification 
based on histidine affinity for divalent metal ions by passage over a nickel column. The 
removal of the approximately 3 kDa N-terminal polyhistidine extension was effected by 
incubation with enterokinase which released the recombinant PLP-C. This polyhistidine 
fragment was assumed to be excluded after dialysis with a 6 kDa membrane. 

The recombinant PLP-C protein isolated from the two bacterial systems had similar 
molecular weight and N-terminal amino acid sequence identity with that of native placental 
PLP-C (Deb et al., 1991b; Ogilvie et al., 1990b). A minor species corresponding to PLP- 
C beginning at residue 7 was also detected in the expression system. Studies describing 
the expression of recombinant rat placental PLP-A in a mainmalian ceU line also identified 
minor species representing N-terminally truncated isoforms of PLP-A (Deb et al., 1993). 
The antiserum generated against recombinant PLP-C demonstrated cross reactivity with the 
25 kDa non-glycosylated and the 29 kDa glycosylated forms of PLP-C in Westem 
immunoblot analysis. 

This study also described the expression of PLP-B as a recombinant protein 
utilizing a mammalian expression vector pMXSND. This vector has previously been 
shown to be effective in the expression of other members of the PRL-GH family from both 
rat and murine sources (Cohick et al., 1997; Colosi et al., 1988; Deb et al., 1993; Lee and 
Nathans, 1988). The choice of mammalian expression was also important based on the 
fact that the native protein has been detected only as a 30 kDa glycosylated species (Ogilvie 
et al, 1990b; Duckworth et al., 1988). The lack of cloning sites within pMXSND 
necessitated that the PLP-B cDNAbe blunt-end ligated into the vector and that transformed 
colonies be screened for the presence of the PLP-B cDNA insert in the correct orientation 



86 

by restriction enzyme digestion prior to transfection into CHOKl cells. Stable transfectants 
were selected after Northern blot analysis using a ^^P-labeled cDNA probe. The 
conditioned medium of stably transfected clonal colonies exhibited the presence of a 30 kDa 
immunoreactive band in Western immunoblot analysis corresponding in molecular weight 
to native placental PLP-B protein. 

This study further described the purification of native preparations of PLP-B and 
PLP-C from placental explant conditioned medium in immunoaffinity chromatography 
using antibodies generated against PLP-B and PLP-C. Purified native PLP-B preparations 
were separated by 2D-SDS polyacrylamide gel electrophoresis and Western immunoblot 
analysis demonstrated the presence of 30 kDa molecular weight species with similar 
molecular weight and pi to that of the native protein (Ogilvie et al., 1990b). Similarly, 
purified native PLP-C preparations showed the presence of several immunoreactive species 
in Western immunoblot analysis as well as on silver stained gels. The presence of both the 
25 kDa non-glycosylated and 29 kDa glycosylated forms of native placental PLP-C has 
been previously characterized (Deb et al., 1991b). The other minor species in these 
preparations may represent products of genes which are closely related to PLP-B and PLP- 
C or, alternatively, are post-translational isoforms of these proteins. 

In summary, PLP-B and PLP-C were both successfully expressed as recombinant 
proteins in mammalian and bacterial systems, respectively. Placental PLP-C is expressed 
as both non-glycosylated and glycosylated species. Our efforts to express PLP-C in 
mammalian systems were not successful. In this regard, other researchers in the field have 
similarly demonstrated an inability to recombinantly express PLP-C in a mammalian system 
(M. Soares, personal communication). Antiserum was generated against PLP-C 
recombinant protein and, along with an antipeptide antiserum against PLP-B, utilized in the 
immunoaffinity purification of the native proteins from placental explant conditioned 
medium. The availability of these protein preparations should allow for their further 
characterization. 



87 



106 



49 



32.5 



28 




hCAU-PLP-C 



18 



Figure 6-1. SDS-PAGE analysis of the purification of hCAII-PLP-C fusion 
protein. AUquots from different steps in the purification of hCAII-PLP-C fusion 
protein were separated on 10% polyacrylamide gels under reducing conditions and 
visualized by Coomassie brilliant blue staining. Lane A, 50 |ig cell lysate prior to 
affinity purification; lane B, 50 |xg cell lysate after affinity purification; lane C, 50 
|ig pAMBS column wash; lane D, 15 p.g affinity purified fusion protein; molecular 
weight standards (x 10^) kDa are indicated on the left. 



BCD 



A B C D 




) ■tf'^i 



.k 



>&i 



hCAII-PLP-C 



Anti-PLP-C 



Anti hCAII 



Figure 6-2. Western immunoblot analysis of hCAII-PLP-C fusion protein 
preparations following each purification step. Samples were separated on 10% 
polyacrylamide gels under reducing conditions and analysed by immunoblotting 
using antisera (1:800) raised against PLP-C oligopeptide (Fig.6-2, left panel) or 
hCAII (Fig. 6-2, right panel). Lane A, 50 |a,g cell lysate prior to affinity 
purification; lane B, 50 |j,g cell lysate after affinity purification; lane C, 50 |xg 
pAMBS column wash; lane D, 15 |j,g affinity purified fusion protein; molecular 
weight standards (x 103) ]^£)a are indicated on the left. 



89 



A B 




18 



Figure 6-3. SDS-PAGE analysis of hCAII-PLP-C fusion protein cleaved by 
enterokinase. The recombinant PLP-C was separated from hCAII by pAMBS 
affinity chromatography, and then concentrated and dialysed. Samples were 
separated on 10% gels under reducing conditions and stained with Coomassie 
brilhant blue. Lane A, 25 |ig enterokinase-cleaved proteins; lane B, 3.5 |i.g 
purified recombinant PLP-C; molecular weight standards (x 10^) kDa are indicated 
on the left. 



90 



Ndel NotI 




Hindi Eael 

hCAII I PLP-C I 



p0304hCAII-PLP-C 



Hindi 



Eael 



3' 



PLP-C 



His-Tag Oligo 



Enterokinase 
cleavage site 



pET22b(+) His-Tag PLP-C 



pET22b(-l-) 



Figure 6-4. Schematic of the construction of a vector expressing PLP-C with an 
N-terminal polyhistidine tract. The top line shows the pET22b(+) plasmid and the 
hCAII-PLP-C vector being cut with restriction enzymes as indicated. The middle 
lines show the three way ligation of pET22b(-i-), an oligo encoding a 9 mer 
polyhistidine tract and the PLP-C cDNA fragment excised from the hCAILPLP-C 
vector. The bottom line shows the structure of the pET22b(-l-)His-PLP-C vector 
and the position of the enterokinase cleavage site at the junction of the histidine 
tract and the PLP-C cDNA insert. 



91 



WT 
Mk 



Ndel Digests 

+ - + - + 



2.2 kb 



550 bp 




Figure 6-5. Ndel diagnostic digests of plasmids from JM109 bacterial colonies 
transformed witli the pET22b(+)His-PLP-C ligation mix. Photograph shows the 
diagnostic cleavage of five plasmids with Ndel for the presence of the PLP-C 
insert. (-) indicates an uncut plasmid, while (+) indicates the plasmid after 
overnight incubation with restriction enzyme Ndel. Plasmid number 2 in the fifth 
lane from the left shows the presence of the expected 700 bp PLP-C insert after 
Ndel digestion. 



Figure 6-6. SDS-PAGE analysis of the steps in the purification of pET22b(+)His- 
PLP-C protein. Aliquots were separated on 10% polyacrylamide gels under 
reducing conditions and visualized by Coomassie brilhant blue staining. (A). Lane 
A, 10 fig cell lysate prior to passage over the nickel affinity column; lane B, 10 |ig 
cell lysate after affinity purification; lane C, 10 fig column wash; lane D, 3 [ig 
affinity purified protein; Lane E, 10 |a.g of the bacterial pellet solubilized in N- 
lauroylsarcosine (detergent), lane F, 10 jig solubihzed after passage over the nickel 
column; lane G, 3 \ig column eluate; lane D, 3 [ig affinity purified protein after 
dialysis. (B). SDS-PAGE of enterokinase cleaved pET22b(-H)His-PLP-C protein. 
The protein was digested with enterokinase (Lane A) and recombinant PLP-C 
separated from the His tag by dialysis (Lane B). Molecular weight standards 
(xl03) are indicated on the left. 



(6A) 



93 



B 



106 



53 



32 
28 



18 






D 



E 



G H 



r^^ 






(6B) 



B 



106 



53 

32 
28 

18 






















94 



EcoRI 



Hindi 



Xhol 



pGEM-3 PLP-B pGEM-3 
EcoRl, Hindu. 

1 



i 



c 



1 



PLP-B 

Klenow, dNTPs 






pMXSND 



pMXSND 



pMXSND 
I Klenow, dNTPs 




PLP-B 





pMXSND 



T4DNA 
Ligase 



pMXSND 

CL\P 

(Dephosphorylation) 





pMXSND 



pMXSND 



pMXSND 



PLP-B 



pMXSND 



Figure 6-7. Schematic of the construction of a mammalian vector expressing PLP- 
B. The top line shows the pMXSND vector and the pGEM3-PLP-B plasmid being 
cut with the relevant restriction enzymes as indicated. The second and third lines 
show the creation of blunt-ended pMXSND and the PLP-B cDNA fragment 
preparatory to Ugation after dephosphorylation of the pMXSND vector. The 
bottom line shows the structure of the pMXSND-PLP-B vector. 



95 



123456789 10 11 






'.^■fe^'l-ta*-' 






2.2 kb 



550 bp 



Figure 6-8. BamHI diagnostic digests of plasmids from JM109 bacterial colonies 
transformed with the pMXSND-PLP-B ligation mix. Photograph shows the 
diagnostic cleavage of eleven plasmids with BamHI for the presence of the PLP-B 
insert in the correct orintation. The plasmid in lane number 7 and 10 from the left 
shows the presence of the PLP-B insert after BamHI digestion. Lane 10 possesses 
the insert in the correct orientation as seen by the expected 1250 bp fragment as 
opposed to the 750 bp fragment in lane 7. 



96 



PLP-B 

clones 




p-Actin 




Figure 6-9. Northern blot analysis of total RNA from clonal colonies of CHOKI 
cells transfected with the pMXSND-PLP-B expression vector. RNA was 
denatured, electrophoresed, transferred onto nylon membranes and hybridized with 
32P-PLP-B. Colonies 4, 5 and 7 were amplified and used for the purification of 
PLP-B from conditioned medium. 



97 



PLP-B (lig) 
Cond. Media 







6 Hg 12 ng 18 ng 



108 




53 



.«# 



28 



PLP-B 



18 



i. 



MwitUili 



Figure 6-10. Western immunoblot analyses of PLP-B levels in the conditioned 
media from stably transfected CHOKl cells. Samples were separated on 10% 
polyacrylamide gels under reducing conditions and analysed by immunoblotting 
using antisera (1:1000) raised against PLP-B oligopeptide Increasing quantities of 
conditioned media from CHOKI cells stably transfected with the pMXSND-PLP-B 
construct. Arrow indicates the location of the PLP-B immunoreactive band. 0, 
represents conditioned medium which had passed through a 10 kDa cutoff 
membrane used in the protein concentrating step. Molecular weight standards (x 
103) are indicated on the left. 



98 



45K 




45 K 



29 K 



20 K 



Native PLP-B 



Figure 6-11. Two dimensional SDS-PAGE analyses of PLP-B preparations. 
Native protein was immunopurified by immunoaffinity chromatography and 
electrophoresed on 2D gels and analysed by silver staining for PLP-B (Figure 6- 
11 A). Samples were run on duplicate gels and electroblotted onto nitrocellulose 
and analysed by Western immunostaining with antisera to PLP-B (Figure 6-1 IB). 
Blots were probed with an antipeptide antiserum against PLP-B at 1 : 1000 dilution, 
followed by incubation with horse-radish peroxidase conjugated with anti-rabbit 
IgG. Bands were visualized by incubation with 3-amino-9-ethylcarbazole in the 
presence of 0.0157o hydrogen peroxide. 



99 



45K 

29K 
20K 

B 

45K 



29K 



20K 




Native PLP-C 



Figure 6-12. Two dimensional SDS-PAGE analysis of PLP-C preparations. 
Native protein was purified by immunoaffinity chromatography and 
electrophoresed on 2D gels and analysed by silver staining for PLP-C (Figure 6- 
12A). Samples were run on duplicate gels and electroblotted onto nitrocellulose 
and analysed by Western immunostaining with antisera to PLP-C (Figure 6-12B). 
Blots were probed with an antiserum against recombinant PLP-C at 1:1000 
dilution, followed by incubation with horse-radish peroxidase conjugated with 
anti-rabbit IgG. Bands were visualized by incubation with 3-amino-9- 
ethylcarbazole in the presence of 0.015% hydrogen peroxide. 



CHAPTER 7 
CHARACTERIZATION OF NATIVE AND RECOMBINANT PROLACTIN-LIKE 

PROTEINS B AND C 



Introduction 

The recombinant proteins PLP-B and PLP-C were expressed and purified as 
described in the previous chapter. In addition, native forms of the PLP-B and PLP-C 
proteins were purified from the conditioned medium of basal zone explant cultures by 
immunoaffinity chromatography. This chapter discusses the evaluation of the biological 
activity of the purified proteins. Both the potential lactogenic and angiogenic activities have 
been evaluated using standard in vitro biological assay systems. 

Firstly, PRL is known to exhibit a broad range of distinct physiological actions 
important in the course of reproduction (Handwerger et al., 1992). The secretion by the 
placenta of high levels of placental lactogens and PRL-related proteins during pregnancy 
has led to the hypothesis that these proteins are of importance in the placental-fetal growth 
axis. Secondly, during the course of gestation, fetal growth and development require 
enhanced placental transport and uterine blood flow which is dependent on placental 
neovascularization (Reynolds et al., 1986). Two members of the placental PRL-GH 
family, proliferin (PLF) and proUferin related protein (PRP), have recently been shown to 
display angiogenic and angiolytic activity, respectively (Jackson et al., 1994). Our 
laboratory has previously studied several animal models for low birth weight and found 
that impaired placental growth and vascular development was correlated with decreased 
secretion of PLP-B and PLP-C proteins by rat placental tissue (Conliffe et al., 1995; 
Shiverick et al., 1991). These observations led us to further investigate the potential for 
these two proteins, PLP-B and PLP-C to exhibit lactogenic and angiogenic activities. 



100 



101 

Results 

Effects of Native PLP-B and PLP-C and Recombinant PLP-C on Nb2 Lymphoma Cell 
Proliferation 

Native PLP-B and PLP-C were evaluated for lactogenic activity utilizing a classic 
cell proliferation bioassay technique (Gout et al., 1980; Tanaka et al., 1980) with Nb2 
lymphoma cells. Ovine PRL was used as a positive control and produced a dose- 
dependent stimulation of ^H-thymidine uptake with an EC,o of approximately 0. 1 nM. 
Both native PLP-B and PLP-C exhibited some lactogenic activity but only at concentrations 
above 100 ng/ml (Figure 7-1). The maximal activity of native PLP-B and PLP-C observed 
was, therefore, at concentrations which were at least 3 orders of magnitude greater than that 
of standard oPRL controls. The two PLP-B and PLP-C preparations were further analyzed 
for the presence of PRL and PL-II by Western immunoblot analysis in order to determine 
whether residual amounts of these known lactogenic placental proteins in the native PLP 
preparations could account for the observed activity. In data not shown, immunoreactive 
bands corresponding to the PL-II or PRL proteins were not observed, nor were these 
bands observed on silver-stained 2D gels of these PLP proteins. By comparison to the 
native preparation, recombinant PLP-C purified from bacterial expression systems did not 
exhibit any lactogenic activity relative to the ovine PRL control in this bioassay (Figure 7- 
2). Thus, evidence suggests that the lactogenic activity expressed by the native PLP-C 
protein preparation may be a consequence of glycosylation which was absent from the 
recombinant protein expressed in bacteria. 

Effects of Placental Conditioned Medium, and Native PLP-B and PLP-C on Endothelial 
Cell Migration 

Gestation day 18 placental tissue was cultured in serum-free medium for 24 hr and 
the conditioned medium evaluated for the ability to stimulate the directional migration of 



102 

human retinal endothelial cells as a measure of angiogenic activity. Medium from the 
cultures of placental (basal zone) tissue markedly stimulated the number of endotheUal 
cells/well migrating through the collagen-coated membranes. The stimulating activity in 
placental conditioned media was highly significant relative to that exhibited by the 10% 
FBS positive controls (Figure 7-3), being 5-7 fold greater than that of the negative controls 
(p < 0.001). In addition, the stimulatory activity of placental conditioned media appears to 
be dose-related in that the response was greater with exposure to 250 [ig compared to 50 
|ig of secreted protein. 

In the cell migration assay, neither native PLP-B nor PLP-C preparations exhibited 
any significant angiogenic activity, even at concentrations up to 5 |J.g/ml (Figure 7-4). 
These data are representative of three experiments with different preparations of native 
PLP-B and PLP-C. The results indicate that day 18 basal zone conditioned medium 
possesses secreted, soluble angiogenic factor(s), whereas native PLP-B and PLP-C 
immunopurified from this media do not exhibit any angiogenic activity as measured by 
chemotactic activity towards endotheUal cells. 

Discussion 

The secretion by the placenta of high levels of placental lactogens and PRL-related 
proteins during the course of pregnancy has led to the hypothesis that they may be of 
importance in the placental-fetal growth axis. Hence there is great interest in determining 
whether or not these newly discovered proteins were "PRL-like" in their ability to bind the 
PRL receptor or to exhibit similar bioactivities. Studies characterizing these rodent 
placental proteins have demonstrated that some of them do in fact exhibit PRL-like activities 
in their ability to produce lactogenic responses in rat Nb2 lymphoma cells (Gout et al., 
1980) and in mammary gland epithelial cell differentiation (Handwerger et al., 1984;, 
Soares et al., 1983; Southard et al., 1986). Mouse and rat PL-I, II and Iv all exhibit such 



103 

activity, and rPL-I has been reported to be even more stimulatory than ovine PRL in the 
Nb2 assay. 

As members of the PRL-GH family, both native PLP-B and PLP-C as weU as 
recombinant PLP-C were evaluated for potential lactogenic bioactivity. The native protein 
preparations exhibited some detectable activity relative to that of ovine PRL in the Nb2 
lymphoma bioassay, but at concentrations which were several orders of magnitude greater 
than that for the EC50 of ovine PRL. In this regard, the native preparations of PLP-B and 
PLP-C were further evaluated for the presence of contaminating residual amounts of PRL 
and PL-II (known lactogenic proteins) by Western immunoblot analysis and silver staining 
(data not shown) of 2D gels. The results, however, were negative for the presence of 
either known lactogenic protein. The presence of measurable lactogenic activity in native 
PLP-C led us to express a recombinant form of the protein for further evaluation of 
biological activity. When analyzed in the Nb2 lymphoma bioassay, however, recombinant 
PLP-C had no mitogenic activity. Furthermore, in data not shown, neither native PLP-B 
nor PLP-C were able to compete effectively for PRL binding to the long form of the PRL- 
receptor, nor were recombinant forms of the two proteins able to compete for binding to the 
rat PRL receptor (K. Shiverick, personal communication). 

An analysis of the potential PLP-B and PLP-C three dimensional structure would 
lend support to these findings. The structure of the PRL-GH family of proteins, based on 
the X-ray crystallographic analysis of GH and mutagenesis studies in PRL, is thought to be 
composed of an up-up-down-down, four a-helical bundle motif, linked by interconnecting 
loops (Abdel-Meguid et al., 1987; de Vos et al., 1992; Goffin et al., 1992, 1993, 1994, 
1996; Somers et al., 1994). As a consequence of the placental PRL-like proteins being 
folded to form this four a helix type structure, the amino acid residues exposed on then- 
surfaces will vary since the helical regions are not highly conserved, with the exception of 
helix 4. This probably allows for variations in the properties available for interaction with 
other macromolecules (Goffin et al., 1996; Southard and Talamantes, 1991), this being true 



104 

for the lactogenic as well as non-lactogenic members of the family. Mutational analysis of 
the lactogenic mouse PL-II (mPL-II), along with sequence comparisons with the other 
members of the placental PRL family, led to the localization of several residues essential for 
maximal receptor binding and bioactivity (Davis and Linzer, 1989). These residues, 
A15I9, AiglTl, Aigl75, Lysl85, Asnl95 on mPRL, map to helix 1 and 4 of the putative 
four a helical folding pattern. 

For the rat placental proteins of unknown biological activity, PLP-B lacks two of 
the key lactogenic residues while PLP-C has none of them. The fact that recombinant PLP- 
C did not exhibit the lactogenic activity observed for the native protein may also be a 
consequence of the lack of glycosylation of the recombinantiy expressed bacterial protein. 
Altematively, native preparations possessed some undetected soluble factor with significant 
lactogenic activity. Croze et al (1990) isolated a cDNA from rat decidua identical to the 
previously identified placental PLP-B cDNA. The expression of decidual PLP-B mRNA 
early in gestation after implantation to midgestation prompted Croze and co-workers (1990) 
to suggest that PLP-B could be a candidate gene which encodes the decidual luteotrophin of 
pregnancy. This protein has been shown to specifically interact with the luteal PRL 
receptor and stimulate the corpus luteum to produce progesterone (Jayatilak et al., 1989). 
The low potency of lactogenic response by native PLP-B and absence of high afiinity 
binding to the PRL receptor (data not shown) is evidence that PLP-B is not the decidual 
luteotrophic protein. 

Jackson et al (1994) has recently demonstrated that the midgestation mouse placenta 
secretes an angiogenic activity that corresponds primarily to PLF, followed by a factor that 
inhibits angiogenesis which is PRE Recombinant PLF and PRP were further found to 
modulate the migration of endothelial cells in vitro and neovascularization in vivo (Jackson 
et al., 1994; Volpert et al., 1996). Previous analysis of the receptor binding activity of 
recombinant PLF showed a specific association with the insuhn-like growth factor 
IFmannose 6-phosphate (M6P) receptor (Lee and Nathans, 1988). More recent data appear 



105 

to indicate that PLF manifests its angiogenic activity as a result of its interaction with this 
receptor (Volpert et al., 1996). Data from our laboratory indicated that both the mRNA and 
protein for placental PLP-B were significantly decreased in a low birth weight rat model 
where reduced placental vascular development was also observed (Conliffe et al., 1995). 
These observations led to the evaluation of native PLP-B and PLP-C for potential 
angiogenic activity in an attempt to elucidate their role in fetal-placental development. 

Our data clearly show that gestation day 18 rat placenta secretes soluble factors 
which significantly stimulate endothelial ceU migration. Our finding that late gestation rat 
placenta expresses predominantly stimulatory factors is in contrast with the report of 
Jackson et al (1994) that conditioned medium from late gestation mouse placental cultures 
was inhibitory to endothelial cell migration. The difference may be a reflection of our use 
of the basal zone (junctional zone) tissue for explant culture as opposed to the whole 
placenta which includes the labyrinth zone (Davies and Glasser, 1968). An additional 
possibility is that endothelial cells isolated from human retina differ from bovine adrenal 
capillaries in their responsiveness to chemotactic factors. In this regard, research on bovine 
models shows that the fetal placental tissue produces factors which inhibit endothelial cell 
migration and proliferation throughout the course of gestation (Reynolds and Redmer, 
1995). Thus some evidence suggests that where these factors may function to limit 
vascular development is within the maternal placental vasculature. 

The present study has also found that PLP-B and PLP-C immunopurified from the 
conditioned media did not show chemotactic activity towards endothelial cells. Thus it 
appears that other soluble angiogenic factors are released by the rat placental explants, 
which may include insuhn-like growth factors (IGF) (Grant et al., 1987, 1992), as well as 
basic fibroblast growth factor (Hondermarck et al., 1990). 

In summary, native PLP-B and PLP-C purified from rat placental conditioned 
media using immunoaffinity chromatography exhibit some lactogenic activity at 
concentrations above 100 ng/ml. In comparison, recombinant PLP-C purified from a 



106 

bacterial expression system shows no significant lactogenic activity in Nb2 lymphoma 
bioassays. Furthermore, neither native PLP-B nor PLP-C appear to be the rat homologues 
for PLF as factors which stimulate neovascularization of the placenta. 



107 



20000- 



•4-) 

a 

u 
o 

O 

u 

St 



H 



15000- 



10000- 



5000- 




T r 

100 1000 



oPRL (ng/ml) 



Figure 7- 1 . Effects of native preparations of PLP-B and PLP-C on Nb2 lymphoma 
cell proliferation. Cells (5 x 10^), were treated in triplicate wells with varying 

concentrations of ovine PRL (D), PLP-B (*) or PLP-C (A) at 37oC for 44 hr. 
Cells were harvested after a 4 hr incubation with -^H-thymidine. Mean values + 
SEM for two experiments are presented. 



108 



120- 



ir> 



g 

2 

© 
a 

u 
o 

s 

a 






1 oPRL 



I 

& 

O 




0.001 



1 1 

1000 10000 



HORMONE CONCENTRATION 

(ng/ml) 



Figure 7-2. Effect of recombinant PLP-C on Nb2 
lymphoma cell proliferation. Cells (8 x 10''^) were treated in 
triplicate with varying concentrations of ovine PRL (•) and 
recombinant PLP-C (♦) at 37° C for 44 hr. 3H-thymidine 
incorporation was determined after a 4 hr labelling period. 
Mean values ± SEM for three experiments are presented. 



109 




^ 



en 



a 


B 


■Si 


■Sh 





s 




o 


p< 


^ 






pa 



Figure 7-3. Quantitation of the angiogenic effect of day 18 placental (basal 
zone) conditioned medium on retinal endothelial cell migration. Details are as 
described in Materials and Methods. Upper wells were loaded with FBS, 
serura-free, or placental conditioned medium. Data are expressed as mean ± 
SEM of the number of migrating cells from three separate experiments. * P< 
0.001 compared to serum-free media (SFM). 



110 





0) 




«PN 




«M 


< 






P. 


^ 


C/J 


^ 


0^ 


s 


• 


PQ 


© 




>^ 


^ 


fl 




c« 




0^ 




1 


1 


1 


S 


■^ 


"ftjD 


"Bh 


■fti 


s 





s 


s 


^ 


00 


V) 


rH 


^ 


e> 


U 


u 


(L 


tt 


On 


Clh 


\- 


a« 


n:i 


h-3 


p^ 


^ 


Ph 


ftn 



Figure 7-4. Quantitation of the angiogenic effect of gestation day 18 placental 
(Basal Zone) conditioned media, native PLP-B and native PLP-C on retinal 
endothelial cell migration. Details are as described in Materials and Methods. 
Upper wells were loaded with BZ medium and PLP-B or PLP-C proteins. 
Data are expressed as mean ± SEM of the number of migrating cells from three 
separate experiments. * P< 0.001 compared to serum-free media (SFM). 



CHAPTER 8 
CONCLUSIONS AND FUTURE DIRECTIONS 



There has been growing public concern about the abiUty of environmental 
contaminants to contribute to adverse effects upon human health. This study grew out of 
the desire to investigate the potential mechanisms by which these agents may play a role in 
placental growth and development, and uterine disease pathologies, with particular regard 
to endometriosis. The objectives of this study were two-fold. Firstly, we investigated the 
cellular and molecular processes which could be altered by B(a)P and TCDD, two AhR 
agonists, in a human endometrial cell line, RL95-2. Secondly, this study attempted to 
characterize the potential role(s) of two placental proteins designated PLP-B and PLP-C 
whose expression has been shown to be altered in low birth weight animal models and by 
exposure to pNF. The conclusions which may be derived from this study, as well as the 
potential directions of future research efforts are described below. 

The first part of this study demonstrated that both TCDD and B(a)P were able to 
induce the expression of members of the cytochrome P450 family, an action classically 
shown to be a consequence of AhR activation (Whitlock, 1993). B(a)P (a major 
contaminant in cigarette smoke), but not TCDD, is able to significantly decrease the 
expression of immunoreactive EOF receptor protein in RL95-2 cell cultures. A down- 
modulation of the EOF receptor has previously been observed after both B(a)P and TCDD 
exposure of human keratinocytes (Hudson et al., 1985). Our results indicate that receptor 
down-modulation is not directly correlated with CYPlAl induction and it is possible that 
reactive metabolites have a role to play in the process, since B(a)P, in contrast to TCDD is 
readily metabolized to a series of reactive intermediates able to bind cellular macromolecules 
(Gelbion 1980; Leadon et al., 1988). 

Ill 



112 

Furthermore, B(a)P, but not TCDD, was able to significantly decrease the 
proliferation of the endometrial adenocarcinoma cells. Endometriosis is an estrogen 
responsive disease, and estrogen actions on the uterus may be mediated through growth 
factors like EGF (Lingham et al., 1988; Nelson et al., 1991). Our data correlate with the 
etiological association of cigarette smoking with a decreased incidence of endometriosis. 
The role of reactive metabolites of B(a)P as a potential mechanism of EGF receptor down- 
modulation should be examined by analysis of RL95-2 cell cultures for metabolic products. 
Furthermore, the use of an antagonist to the AhR would also aid in determining whether or 
not the effects observed on the EGF receptor and/or the formation of B(a)P metabolites are 
mediated as a result of receptor activation. 

This study next evaluated the potential role of genes and gene products which could 
contribute to the elaboration of changes in the invasive phenotype of these endometrial cells 
as a mechanism of disease promotion. TCDD, but not B(a)P, was observed to increase the 
steady state levels of uPA mRNA. Neither compound, however, was able to significantly 
alter the fibrinolytic activity of the conditioned medium of treated cells, as evaluated by 
fibrin zymography. Similarly, both compounds were able to increase the expression of 
TIMP-1 mRNA, but were without effect on TIMP-2 expression levels. 

B(a)P, but not TCDD, was able to significantly decrease the overall ability of 
treated cells to invade matrigel membranes. The fact that cellular invasiveness was 
decreased by B(a)P correlates with epidemiological data regarding this disorder, however, 
the TIMP-l data from our study does not. Our interpretation of the invasion data may need 
to take into consideration the experimental design of the assay in which the cells invade 
through an inverted membrane system. As a consequence of this, the observed inability of 
B(a)P treated cells to attach to the matrigel membrane would account for the observed 
B(a)P invasion data, an outcome which brings into consideration the role of adhesion 
factors in the invasion process. Thus an investigation of the B(a)P effects on potential 
alteration in the expression of members of the integrin and cadherin family may be 



113 

warranted, presently an area of research which may be gaining momentum in the field of 
endometriosis (Lessey et al., 1994; van der Linden et al., 1994). The fact that B(a)P 
resulted in a significant growth arrest of our endometrial cell cultures may also contribute to 
the observation of reduced invasive activity by these cells. Finally, the overall invasiveness 
of these cells may be the net result of alterations in the expression of other members of the 
MMP and TIMP families. An investigation of the potential changes in the expression of 
these genes and gene products would contribute to a better understanding of the control of 
the invasion process in uterine cells. 

This part of the study also demonstrated that TCDD was able to significantly 
increase the expression of IL-lp and TNF-a, cytokines which potentially could contribute 
to the proinflammatory processes observed in endometriosis (Koyama et al., 1993; Ramey 
et al., 1993; Rana et al., 1996). The time course of TCDD induction of these responses 
may be important in that IL-lp has been shown to increase the secretion of TNFa from 
cytotrophoblast cell cultures, albeit without a change in mRNA levels for TNFa (Knofler et 
al., 1997). These genes are regulated at a number of levels, especially at the post- 
transcriptional level in the case of TNF-a (Tabibzadeh, 1991). Thus it would be 
worthwhile to examine whether the observed changes in mRNA are manifested as 
increased levels of protein expression for these cytokines. 

The fact that TNFa has been shown to alter the ability of cells from primary 
endometrial cultures to attach to basement mesothehal cells (Zhang et al., 1993b), as well 
as regulate the dyscohesion of epithehal cells (Tabibzadeh et al., 1995), may indicate a role 
for this cytokine in the modulation of adhesion factor expression. Furthermore, the 
observation of increased levels of prostaglandins in the peritoneal fluid of endometriotic 
patients may be the result of cytokine mediation. Prostaglandins have been implicated in 
the increase in endometrial vascular permeability which occurs during the implantation 
process (Chen et al., 1995). The potential of TCDD and B(a)P treated RL95-2 cultures to 



114 

release prostaglandins as a consequence of enhanced cytokine production can also be 
investigated. 

The alterations produced by TCDD and B(a)P treatment of the RL95-2 endometrial 
carcinoma cells are summarized in Figure 8-1. As outlined, TCDD tends to stimulate what 
could be chai-acterized as proinflammatory responses in these cells, while B(a)P produces 
decreased growth and decreased invasive potential. Further work, however, needs to be 
done to more completely characterize the overall responses of these cells to these 
prototypical environmental agents. 

The second part of this study attempted to characterize the biological activities of 
two proteins secreted by the rat placenta during pregnancy. PLP-B and PLP-C were 
successfully purified from the conditioned medium of placental explant cultures using 
antibodies generated against the proteins for immunoaffinity chromatography. The native 
proteins exhibited molecular weights, pi and immunoreactivity similar to that of the 
previously characterized placental proteins (Deb et al., 1991b; Ogilvie et al., 1990b). Both 
PLP-B and PLP-C were also expressed as recombinant proteins in mammalian and 
bacterial expression systems, respectively. This is the first successful expression of 
recombinant PLP-C since attempts to express it in mammalian host systems have been 
unsuccessful. 

Evaluation of the biological activity of the native PLP-B and PLP-C preparations 
demonstrated that both protein preparations exhibited low levels of lactogenic activity; 
however, this lactogenic activity was not observed for the recombinant PLP-C. The lack of 
activity by recombinant PLP-C may be a consequence of glycosylation, but this is presently 
difficult to determine due to the inability to express PLP-C in mammaUan host systems. 
The placental conditioned medium, but not native PLP-B or PLP-C, exhibited significant 
angiogenic activity as evaluated by the ability of these preparations to stimulate the 
chemotaxis of human retinal endotheUal cells. Future studies could utilize the purified 
recombinant proteins in order to localize their binding sites after ['"^I] labeling. 



115 

Crosslinking studies could be performed with [^^^I] labeled probe in an attempt to 
chai"acterize the receptors for these proteins. The fact that PLP-B and PLP-C are major 
secretory placental proteins present during the second half of pregnancy argues for a 
physiological role during this period. 



116 



Epidemiology 




CIGARETTE SMOKING 


DIOXIN 


, , Endometriosis 


Endometriosis 


Gene Expression 




B(a)P 


TCDD 


i Proliferation 


IL-ip 


Iegfr 


TNF-a 


Invasion/Attacliment 


uPA 


|timp-i 


TIMP-1 


Phenotvpe 




OVERALL 


OVERALL 


Proliferation 
i Invasion/Attachment 


^ Proinflammatory 
Responses 



Figure 8-1. Comparison of the epidemiological factors in the incidence of endometriosis 
with alterations in gene expression in the endometrial cell line RL95-2. 



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BIOGRAPHICAL SKETCH 

Grantley Dexter Charles was born on December 15th 1967, in San Fernando, 
Trinidad. Despite being raised Baptist he spent his formative middle and high school under 
the tutelage of Irish Catholic monks (Presentation Brotherhood) at a parochial Catholic 
Grammar School. Upon graduation he worked as a substitute elementary school teacher 
for a year before entering the University of the West Indies in St. Augustine, Trinidad in 
1987. There, after a three year travail, he received his B.Sc. degree in the Natural Sciences 
with a major in Chemistry. After a one semester stint as a middle and high school teacher, 
he came to the United States in January 1991, to pursue his then dream of a Master's 
Degree in Forensic Science at the John Jay College of Criminal Justice (CUNY) in New 
York. His interest being stirred by drug pharmacology and toxicology, he apphed to and 
was accepted into the Department of Pharmacology and Therapeutics at the University of 
Florida in Fall 1992. There he gravitated to the laboratory of Dr. Kathleen Shiverick where 
for the course of his graduate study he has worked in the area of reproductive and 
developmental toxicology. He has accepted a postdoctoral position at The Dow Chemical 
Company in Midland, Michigan, in the laboratory of Dr. Edward Carney, Project Leader 
for the Developmental and Reproductive Toxicology (DART) Group. 



136 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 



Kathleen T. Shiverick, Chair 
Professor of Pharmacology 
and Therapeutics 

I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 



David N. Silverman 
Professor of Pharmacology 
and Therapeutics 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 




Thomas C. Rowe 
Associate Professor of Pharmacology 
and Therapeutics 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 



and 






4 i V '■ '' 

lefftey/K. Harrison 

Assistant Professor of Pharmacology 

Therapeutics 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope an^-qualily^as a^ 
dissertation for the degree of Doctor of Philosophy. 





William C. Buhi 
Professor of Biochemistry 
and Molecular Biology 



This dissertation was submitted to the Graduate Faculty of the College of Medicine 
and to the Graduate School and was accepted as partial fulfillment of the requirements for 
the degree of Doctor of Philosophy. 



December 1997 




' Dean, College of Medicine 



rf(j 



Dean, Graduate School